ES2382357T3 - Myeloid-type cells transfected for the treatment of retinopathy of prematurity and related diseases - Google Patents

Abstract

The present invention provides the use of cells from a vasculotrophic lineage negative hematopoietic stem cell population for the preparation of a pharmaceutical composition for treating a mammal suffering from or at risk of developing retinopathy of prematurity or a related retinopathic disease wherein the cells are transfected with a therapeutically useful gene encoding for an angiostatic fragment of Trp-RS and wherein the pharmaceutical composition is for administering to the retina of the mammal an amount of cells from a vasculotrophic lineage negative hematopoietic stem cell population effective to promote beneficial physiological revascularization of damaged areas of the retina and to ameliorate damage to the retina caused by the disease.

Description

Myeloid-type cells transfected for the treatment of retinopathy of prematurity and related diseases.

Cross reference to related requests

The present application claims the rights of the US provisional patent application with serial number 60 / 656.122, filed on February 24, 2005.

Government Declaration of Interest

A part of the work described herein was funded by grants number EY11254 and EY12598 from the National Eye Institute of the National Institutes of Health ("National Institutes of Health"). The United States government has certain rights in this invention.

Field of the Invention

The technical field of the invention relates to the treatment of retinopathic diseases. More particularly, it refers to the treatment of retinopathic diseases such as retinopathy of prematurity by administering cells as specified herein to the eye of a mammal that suffers or is at risk of developing such diseases.

Background of the invention

Vascular retinal diseases, including diabetic retinopathy, exudative age-related macular degeneration (ARMD), prematurity retinopathy (ROP) and vascular occlusions, are important causes of visual impairment and blindness. This group of diseases is the center of intense research aimed at identifying new treatment modalities that will help prevent or modify pathological ocular neovascularization. For example, ARMD affects 12-15 million Americans over the age of 65 and causes visual loss in 10-15% of them as a direct effect of choroidal (subretinal) neovascularization. The leading cause of visual loss for Americans below the age of 65 is diabetes; 16 million individuals in the United States are diabetic and 40,000 a year suffer from ocular complications of the disease, often a result of retinal neovascularization. Although laser photocoagulation has been effective in preventing severe visual loss in subgroups of high-risk diabetic patients, the overall incidence at 10 years of retinopathy remains substantially unchanged. For patients with choroidal neovascularization due to ARMD or inflammatory eye disease such as ocular histoplasmosis, photocoagulation, with few exceptions, is not effective in preventing visual loss. Although recently developed non-destructive photodynamic therapies hold the promise of reducing individual loss in patients with choroidal neovascularization not previously treatable, only 61.4% of patients treated every 3-4 months had improved or stabilized vision compared to 45.9% of the placebo group.

Age-related macular degeneration and diabetic retinopathy are the main causes of visual loss in industrialized nations and is thus a result of abnormal retinal neovascularization. Since the retina consists of well-defined layers of neuronal, glial and vascular elements, relatively small alterations such as those observed in vascular proliferation or edema can lead to a significant loss of visual function. Inherited retinal degenerations, such as retinitis pigmentosa (RP), are also associated with vascular abnormalities, such as arteriolar narrowing and vascular atrophy. Although significant progress has been made in identifying factors that promote and inhibit angiogenesis, no treatment is currently available to specifically treat ocular vascular disease.

Inherited retinal degenerations affect as many as 1 in 3500 individuals and are characterized by progressive night blindness, loss of visual field, optic nerve atrophy, arteriolar attenuation, altered vascular permeability and loss of central vision that often progresses to blindness complete (Heckenlively, JR, editor, 1988; Retinitis Pigmentosa, Philadelphia: JB Lippincott Co.). The molecular genetic analysis of these diseases has identified mutations in more than 110 different genes that represent only a small percentage of known affected individuals (Humphries et al., 1992, Science 256: 804-808; Farrar et al. 2002, EMBO J .21: 857-864.). Many of these mutations are associated with enzymatic and structural components of the phototransduction machinery including rhodopsin, cGMP phosphodiesterease, rDS periferin and RPE65. Despite these observations, there is still no effective treatment to slow down or reverse the progression of these degenerative retinal diseases. Recent advances in gene therapy have led to a satisfactory reversal of the rds (Ali et al. 2000, Nat. Genet. 25: 306-310) and rd (Takahashi et al. 1999, J. Virol. 73: 7812-7816 ) in mice and of the RPE65 phenotype in dogs (Acland et al. 2001, Nat. Genet. 28: 92-95) when the wild-type transgene is supplied to photoreceptors or retinal pigmented epithelium (RPE) in animals with a specific mutation

Angiogenesis is the process by which new blood vessels form. In response to specific chemical signals, capillaries sprout from existing vessels, eventually growing in size as the body needs. Initially, the endothelial cells, which line the blood vessels, divide in an orthogonal direction to the existing vessel, forming a solid bud. Then, adjacent endothelial cells form large vacuoles and the cells are rearranged so that the vacuoles themselves are oriented end to end and finally fuse to form the light of a new capillary (tube formation).

Angiogenesis is stimulated by various conditions, such as in response to a wound, and accompanies virtually all tissue growth in vertebrate organisms such as mammals. Angiogenesis also plays a role in certain disease states such as diabetic retinopathy and certain cancers. Tumor growth, for example, requires the growth of blood vessels to provide oxygen and nutrients to the growing tumor tissue.

Angiogenesis can be stopped or inhibited by interfering with the chemical signals that stimulate the angiogenic process. For example, angiogenic endothelial cells produce proteases to digest the basal lamina that surrounds blood vessels, thus opening a path for the new capillary. The inhibition of these proteases, or their formation, can prevent the formation of new vessels. Also, endothelial cells proliferate in response to chemical signals. Particularly important proliferation signals include the families of vascular endothelial growth factor (VEGF) proteins and fibroblast growth factor (FGF). VEGF has been shown to be involved in the vascularization of certain tumors. Interference with these proliferation signaling processes can also inhibit angiogenesis.

Several factors are involved in angiogenesis. Both acidic and basic fibroblast growth factor molecules are mitogenic for endothelial cells and other cell types. A highly selective mitogen for vascular endothelial cells is vascular endothelial growth factor (VEGF).

In the normal adult, angiogenesis is strictly regulated, and is limited to wound healing, pregnancy and the uterine cycle. Angiogenesis is activated by specific angiogenic molecules such as acid and basic fibroblast growth factor (FGF), VEGF, angiogenin, transforming growth factor (TGF), tumor necrosis factor-a (TNF-a) and derived growth factor of platelets (PDGF). Angiogenesis can be suppressed by inhibitory molecules such as interferon-a, thrombospondin-1, angiostatin and endostatin. It is the balance of these naturally occurring stimulators and inhibitors that controls the quiescent capillary vasculature normally. When this balance is disturbed, as in certain pathological states, proliferation, migration and ultimately differentiation of capillary endothelial cells is induced.

Angiogenesis plays a central role in a variety of diseases including cancer and ocular neovascularization. It has also been shown that sustained growth and metastasis of a variety of tumors depend on the growth of new host blood vessels within the tumor in response to angiogenic factors derived from the tumor. Proliferation of new blood vessels in response to a variety of stimuli appears as the dominant finding in most eye diseases that produce blindness including proliferative diabetic retinopathy, ARMD, rubeotic glaucoma, interstitial keratitis and retinopathy of prematurity. In these diseases, tissue damage can stimulate the release of angiogenic factors that result in hair proliferation. VEGF plays a dominant role in neovascularization of the iris and neovascular retinopathies. Although the reports clearly show a correlation between intraocular VEGF levels and ischemic retinopathic ocular neovascularization, FGF probably plays a role. It is known that acidic and basic FGF are present in the normal adult retina, even when detectable levels do not systematically correlate with neovascularization. This may be largely due to the fact that FGF binds very strongly to charged components of the extracellular matrix and may not be readily available in a freely diffusible form that would be detected by conventional intraocular fluid assays.

A final common route in the angiogenic response involves integrin-mediated exchange of information between a proliferating vascular endothelial cell and the extracellular matrix. This class of adhesion receptors, called integrins, are expressed as heterodimers that have a subunit a and 1 in all cells. An integrin of this type, av13, is the most promiscuous member of this family and allows endothelial cells to interact with a wide variety of extracellular matrix components. Peptide and antibody antagonists of this integrin inhibit angiogenesis by selectively inducing apoptosis of proliferating vascular endothelial cells. There are two cytokine-dependent angiogenesis pathways and can be defined by their dependence on different vascular cell integrins, av13 and av15. Specifically, angiogenesis induced by basic FGF and VEGF depends on the integrin av13 and av15, respectively, since antibody antagonists of each integrin selectively block one of these angiogenic pathways in chicken chorioallantoic membrane (CAM) and rabbit corneal membrane models . Peptide antagonists that block all av integrins inhibit angiogenesis stimulated by FGF and VEGF. Although normal human eye blood vessels do not have any integrin, av13 and av15 integrins selectively occur in blood vessels in tissues of patients with active eye neovascular disease. Although only av13 was systematically observed in tissue of patients with ARMD, both av13 and av15 were present in tissues of patients with proliferative diabetic retinopathy. Integrin peptide antagonists administered systemically blocked the formation of new blood vessels in a mouse model of retinal vasculogenesis.

Evidence of possible treatments for retinal neovascular diseases has been greatly facilitated by the development of oxygen-induced retinopathy (OIR) models in several animal species, including cat puppies, beagle, rat and mouse puppies. In each of these models, exposure of newborn animals to hyperoxia (or to alternate hyperoxia and hypoxia) causes regression or retardation of retinal vascular development, followed by abnormal angiogenesis after its return to normal oxygen levels. These models mimic the events that occur during retinopathy of prematurity (ROP), a condition that involves pathological neovascularization that can affect premature infants.

Over the past decade, the OIR mouse model has become the most common model for studying the anomalous angiogenesis associated with oxygen-induced retinopathies. The pattern of vascular anomalies in this model differs slightly from that observed in ROP; in the mouse, the central, posterior retina becomes avascular after exposure to hyperoxia while in human infants the periphery is avascular. However, this is a well accepted model to study the mechanisms of the disease and the possible treatment of hypoxia-induced retinopathy, and vascular changes are very constant, reproducible and quantifiable. In recent years, the use of this model has been extended to the general study of ischemic vasculopathies and related antiangiogenic interventions, and is now widely used in both basic and applied research settings.

The historically common procedure to quantify the proliferative neovascular response in the mouse IOR model is based on the count of the number of cells associated with new vessels that extend from the retina into the vitreous humor (“pre-internal limiting membrane nuclei ( ILM) ”). This is done in sagittal cross sections, usually in regions near (but not including) the optical disc. The procedure is very laborious, time-consuming and plagued with difficulties, including the need to differentiate cells from anomalous vessels from those of hialoid vessels in the vitreous humor. In general, because only every 30th section is evaluated in series, a large part of the tissue is not quantified, possibly introducing large sampling errors. In addition, since complete eyes are immediately sectioned, the evaluation in the same eye of another important parameter of this model is prevented, specifically the degree of vascular obliteration and the rate of retinal revascularization that occurs concomitantly with the formation of abnormal neovascular glomeruli. This parameter is best evaluated in microscopic preparations of complete retinal retina.

For many years, it has been known that there is a population of stem cells in the normal adult circulation and bone marrow. Different subpopulations of these cells can be differentiated into positive (Lin +) or negative (Lin-) hematopoietic lineages. In addition, it has recently been shown that the population of hematopoietic stem-negative stem cells (HSC) contains endothelial progenitor cells (EPC) that can form blood vessels in vitro and in vivo (see Asahara et al. 1997, Science 275: 964-7 ). These cells can participate in normal and pathological postnatal angiogenesis (see Lyden et al. 2001 Nat. Med. 7, 1194-201; Kalka et al. 2000, Proc. Natl. Acad. Sci. USA 97: 3422-7; and Kocher et al. 2001, Nat. Med. 7: 430-6) as well as differentiate into a variety of non-endothelial cell types including hepatocytes (see Lagasse et al. 2000, Nat. Med. 6: 122934), microglia (see Priller et al. 2002 Nat. Med. 7: 1356-61), cardiomyocytes (see Orlic et al. 2001, Proc. Natl. Acad Sci. USA 98: 10344-9) and epithelium (see Lyden et al. 2001, Nat Med. 7: 1194-1201). Although these cells have been used in several experimental models of angiogenesis, the mechanism of addressing EPC to the neovasculature is unknown and no strategy has been identified that effectively increases the number of cells that contribute to a particular vasculature.

Currently, hematopoietic bone marrow stem cells are the only type of stem cells commonly used for therapeutic applications. Bone marrow HSCs have been used in transplants for more than 40 years. Currently, advanced procedures for collecting purified stem cells to develop therapies for the treatment of leukemia, lymphoma and inherited blood disorders are under investigation. Clinical applications of stem cells in humans have been investigated for the treatment of diabetes and advanced kidney cancer in limited numbers of human patients.

WO2004 / 010959 discloses the use of hematopoietic stem-negative stem cells (Lin-HSC) derived from bone marrow and transfected with angiostatic TrpRS fragments to prepare a retinopathy drug.

Summary of the invention

The present invention provides a use of cells in the preparation of a pharmaceutical composition for treating a mammal that is at risk of developing or suffering from a retinopathic disease, in particular a retinopathy of prematurity, in which:

(to)

the cells are a population of myeloid-type bone marrow cells, vasculotropic, of negative lineage, isolated in which the cells express the CD44 and CD11b antigens;

(b)

the cells have been transfected with a nucleic acid encoding an angiostatic fragment of tryptophanyl-tRNA synthetase (Trp-RS);

(C)

The pharmaceutical composition is for the administration of said cells to the mammalian retina in an amount effective to favor the physiological revascularization of damaged areas of the retina and to improve the retinal injury caused by the disease.

Preferably, the mammal is a human patient. In a preferred embodiment, the cells comprise hematopoietic stem cells and endothelial progenitor cells.

Preferably, the cells are autologous to the individual mammal being treated. Preferably, the pharmaceutical composition is for the administration of the cells by intraocular injection. In a preferred embodiment, the pharmaceutical composition is for the administration of the cells to a mammal suffering from retinopathy of prematurity (ROP), such as a human infant, during early stages of the disease. In another preferred embodiment, the pharmaceutical composition is for the administration of the cells administered to a mammal that runs the risk of developing ROP or a related retinopathic state, as a prophylactic agent, before the onset of disease symptoms or before of an infant's exposure to hyperoxia.

The results of the model of oxygen-induced retinopathy (OIR) of ROP indicate that the treatment described herein promotes scarring and vascular recovery in the retina of a mammal suffering from retinopathic disease. In addition, the treatment promotes the recovery of visual neurons due to the neurotrophic effects of the cells. Beneficially, the cells used according to the invention are incorporated into the retinal vasculature and differentiate into endothelial cells, while at the same time they are incorporated into the neural network and improve the degeneration of neuronal cells, such as cone cells in the retina. The cells include cells that selectively select as retinal astrocytes when they are injected intravitreally into the eye, and remain stably incorporated into the neovasculature and the neural network of the eye.

A particular advantage of the eye treatments described herein is a vasculotrophic and neurotrophic rescue effect observed in eyes treated intravitreally with the cells used according to the invention. Retinal neurons and photoreceptors, particularly cones, are preserved and some degree of visual function can be maintained in eyes treated with cells.

Preferably, the diseased retina to be treated according to the invention includes activated astrocytes. This can be achieved by early treatment of the eye with the cells used according to the invention when there is an associated gliosis, or by using a laser to stimulate local proliferation of activated astrocytes.

Brief description of the drawings

In the drawings:

Figure 1 depicts schematic diagrams of developing mouse retina. (a) Development of primary plexus. (b) The second phase of retinal vessel formation. GCL, ganglion cell layer; IPL, internal plexus layer; INL, internal nuclear layer; OPL, external plexus layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium; ON, optic nerve; P, periphery. Panel (c) represents the flow cytometry characterization of separate Lin + HSC and Lin-HSC cells derived from the bone marrow. Top row: Distribution of dot plot cells not labeled with antibody, in which R1 defines the quantifiable-activated area of positive PE staining; R2 indicates positive for GFP; central row: Lin-HSC cells (C57B / 6) and lower row: Lin + HSC cells (C57B / 6), each cell line labeled with PE-conjugated antibodies for Sca-1, c-kit, Flk-1 / KDR, CD31 Tie-2 data were obtained from Tie-2-GFP mice. The percentages indicate the percentage of positively labeled cells of the total Lin-HSC or Lin + HSC population.

Figure 2 depicts the Lin-HSC graft in developing mouse retina. (a) Four days after injection (P6) eGFP + Lin-HSC cells injected intravitreally join and differentiate in the retina. (b) Lin-HSC (B6.129S7-Gtrosa26 mice, stained with 1-gal antibody) establish themselves above the vasculature stained with antibody against collagen IV (the asterisk indicates the tip of the vasculature). (c) Most Lin + HSC cells (eGFP +) four days after injection (P6) were unable to differentiate. (d) murine EC mesenteric eGFP + four days after injection (P6). (e) Lin-HSC (eGFP +) injected into the eyes of adult mice. (f) Image at a few magnifications of eGFP + Lin-HSC (arrows) addressing and differentiating along the pre-existing astrocytic mold in the GFAP-GFP transgenic mouse. (g) Image at further increases in the association between Lin- cells (eGFP) and underlying astrocytes (arrows). (h) GFAP-GFP transgenic control not injected. (i) Four days after injection (P6), eGFP + Lin-HSC migrate to and experience differentiation in the area of the future deep plexus. The figure on the left captures the activity of Lin-HSC in a microscopic preparation of complete retina; The figure on the right indicates the location of the Lin- cells (arrows) in the retina (the upper part is the vitreous side, the lower part is the scleral side). (j) Double labeling with a-CD31-PE and a-GFP-alexa 488 antibodies. Seven days after injection, the injected Lin-HSCs (eGFP, red) were incorporated into the vasculature (CD31). The arrowheads indicate the built-in areas. (k) eGFP + Lin-HSC cells form vessels fourteen days after injection (P17). (l and m) Intracardiac injection of rhodamine-dextran indicates that the vessels are intact and are functional in both the primary (I) and deep (m) plexus.

Figure 3 shows that eGFP + Lin-HSC cells are directed to gliosis (indicated by astrocytes expressing GFAP, image from the far left) induced by both laser (a) and mechanical induced lesion (b) in the adult retina (asterisk) indicates the injured site). The images on the far right are increasing, demonstrating the close association of Lin-HSC and astrocytes. Calibration bar = 20! M.

Figure 4 shows that Lin-HSC cells rescue the mouse vasculature with degeneration of the retina. (a-d) Retinas at 27 days after injection (P33) with collagen IV staining; (a) and (b), retinas that are injected with Lin + HSC cells (Balb / c) showed no differences in vasculature with respect to normal FVB mice; (c) and (d) retinas injected with Lin-HSC (Balb / c) showed a rich vascular network analogous to a wild-type mouse; (a) and (c), frozen sections of the entire retina (the upper part is the vitreous side, the lower part is the scleral side) with DAPI staining; (b) and (d), deep plexus of the complete microscopic retinal preparation; (e) bar graph illustrating the increase in vascularity of the deep vascular plexus formed in retinas in which Lin-HSC cells are injected (n = 6). The degree of deep retinal vascularization was quantified by calculating the total length of vessels within each image. The average total length of vessels / field of large increase (in micrometers) for Lin-HSC, Lin + HSC or control retinas was compared. (f) Comparison of the length of the deep vascular plexus after injection with Lin-HSC (R, right eye) or Lin + HSC (L, left eye) cells from an rd / rd mouse. The results of six independent mice are shown (each color represents a separate mouse).

(g)

and (h) Lin-HSC cells also rescued (Balb / c) the rd / rd vasculature when injected into P15 eyes. It shows the intermediate and deep vascular plexus of retinas in which Lin-HSC (G) or Lin + HSC cells are injected

(H)

 (one month after the injection).

Figure 5 depicts photomicrographs of mouse retinal tissue: (a) deep layer of the complete microscopic retinal preparation (mouse rd / rd), five days after injection (P11) with visible eGFP + Lin-HSC (gray). (b) and (c) P60 retinal vasculature of Tie-2-GFP (rd / rd) mice that received Lin- (b) Balb / c cells or injection with Lin + HSC (c) cells in P6. Only endogenous endothelial cells (stained with GFP) are visible on the left panels of

(b)

 and (c). The central panels of (b) and (c) are stained with antibody against CD31; the arrows indicate the vessels stained with CD31 but not with GFP, the right panels of (b) and (c) show staining with both GFP and CD31. (d) Staining with re-a-SMA to which Lin-HSC (left panel) and control (right panel) were injected.

Figure 6 shows that Lin-HSC transfected with T2-TrpRS inhibits the development of the mouse retinal vasculature. (a) Schematic representation of human TrpRS, T2-TrpRS and T2-TrpRS with an Igk signal sequence at the amino terminal end. (b) Retinas injected with Lin-HSC transfected with T2-TrpRS express the T2-TrpRS protein in vivo. (1) Recombinant T2-TrpRS produced in E. coli; (2) Recombinant T2-TrpRS produced in E. coli; (3) Recombinant T2-TrpRS produced in E. coli; (4) control retina; (5) retinal injected Lin-HSC + pSecTag2A (vector only); (6) retinal injected Lin-HSC + pKLe135 (Igk-T2-TrpRS in pSecTag). (A) Endogenous TrpRS. (B) Recombinant T2-TrpRS. (C) T2-TrpRS of the retina in which Lin-HSC was injected. (c-f) Primary (superficial) and secondary (deep) plexuses representative of injected retinas, seven days after injection; (c) and (d) Eyes injected with Lin-HSC transfected with empty plasmid developed normally; (e) and (f) most of the eyes injected with Lin-HSC transfected with T2-TrpRS showed deep plexus inhibition; (c) and (e) primary (superficial) plexus; (d) and (f) secondary (deep) plexus). The faint outline of the vessels observed in (f) are "filtration" images of the vessels of the primary network shown in (e).

Figure 7 shows the DNA sequence encoding T2-TrpRS with His6 tail, SEQ ID NO: 1.

Figure 8 shows the amino acid sequence of T2-TrpRS with His6 tail, SEQ ID NO: 2.

Figure 9 illustrates photomicrographs and electroretinograms (ERG) of retinas of mice into whose eyes the Lin-HSC and Lin + HSC (controls) were injected.

Figure 10 represents statistical graphs showing a correlation between neuronal rescue (y axis) and vascular rescue (x axis) for both intermediate (Int.) And deep vascular layers of rd / rd mice treated with Lin-HSC .

Figure 11 represents statistical graphs that show no correlation between neuronal rescue (y axis) and vascular rescue (x axis) for eyes of rd / rd mice that were treated with Lin + HSC.

Figure 12 is a bar graph of vascular length (y axis) in arbitrary relative units for eyes of rd / rd mice treated with Lin-HSC (dark bars) and eyes of untreated rd / rd mice (light bars) to the time points of 1 month (1M), 2 months (2M) and 6 months (6M) after injection.

Figure 13 includes three bar graphs of the number of nuclei in the outer neural layer (ONR) of rd / rd mice at 1 month (1M), 2 months (2M) and 6 months (6M) after injection, and demonstrates significant increase in the number of nuclei for eyes treated with Lin-HSC (dark bars) in relation to control eyes treated with Lin + HSC (light bars).

Figure 14 depicts graphs of the number of nuclei in the external neural layer for individual rd / rd mice, comparing the right eye (R, treated with Lin-HSC) in relation to the left eye (L, control eye treated with Lin + HSC) to the time points (after injection) of 1 month (1M), 2 months (2M) and 6 months (6M); Each line in a given chart compares the eyes of an individual mouse.

Figure 15 depicts retinal vasculature and neural cell changes in rd1 / rd1 (C3H / HeJ, left panels) or wild type (C57BL / 6, right panels) mice. The retinal vasculature of the intermediate (upper panels) or deep vascular plexuses (central panels) in microscopic preparations of complete retinas (red: collagen IV, green: CD31) and sections (red: DAPI, green: CD31, lower panels) are shown. of the same retinas (P: day after birth). (GCL: ganglion cell layer, INL: internal nuclear layer, ONL: external nuclear layer).

Figure 16 shows that the injection of Lin-HSC rescues the degeneration of neural cells in rd1 / rd1 mice. (A, B and C), retinal vasculature of the intermediate (Int.) Or deep plexus and sections of the eye injected with Lin-HSC (right panels) and eye injected with contralateral control cells (CD31-) ( left panels) to P30 (A), P60 (B) and P180 (C). (D), the average of the total length of the vasculature (+ or - the standard error of the mean) in retinas in which Lin-HSC or control cells (CD31-) were injected at P30 (left, n = 10 ), P60 (medium, n = 10) and P180 (right, n = 6). The data of the intermediate (Int.) And deep vascular plexus are shown separately (Y axis: relative length of the vasculature). (E), the average numbers of cell nuclei in the ONL at P30 (left, n = 10), P60 (middle, n = 10) or P180 (right, n = 6) of retinas to which control cells were injected (CD31-) or Lin-HSC (Y axis: relative number of cell nuclei in the ONL). (F), Linear correlations between the length of the vasculature (X axis) and the number of cell nuclei in the ONL (Y axis) at P30 (left), P60 (middle) and P180 (right) of retinas at which they were injected with Lin-HSC or control cells.

Figure 17 demonstrates that the function of the retina is rescued by injection of Lin-HSC. Electroretinographic records (ERG) were used to measure the function of retinas in which Lin-HSC or control cells (CD31-) were injected. (A and B), representative cases of retinas rescued and not rescued 2 months after injection. Sections of right eye retina are shown injected with Lin-HSC (A) and left eye injected with CD31- (B) control cells of the same animal (green: vasculature stained with CD31, red: nuclei stained with DAPI). (C), ERG results of the same animal shown in (A) and (B).

Figure 18 shows that a population of human bone marrow cells can rescue degenerating retinas in the mouse rd1 (AC). The rescue is also observed in another model of retinal degeneration, rd10 (D-K). A, human Lin-HSC (hLin-HSC) labeled with green dye can differentiate into retinal vascular cells after intravitreal injection in C3SnSmn.CB17-Prkdc SCID mice. (B and C), retinal vasculature (left panels; upper: intermediate plexus, lower: deep plexus) and neural cells (right panel) in eye injected with hLin-HSC (B) or contralateral control eye (C) 1 , 5 months after injection. (DK), rescue of rd10 mice using Lin-HSC (injected into P6). Retinas representative of P21 (D: Lin-HSC, H: control cells), P30 (E: Lin-HSC, I: control cells), P60 (F: Lin-HSC, J: control cells) and P105 (G are shown : Lin-HSC, K: control cells) (the treated and control eyes are of the same animal at each time point). The retinal vasculature was stained (the upper image in each panel is the intermediate plexus; the central image in each panel is the deep plexus) with CD31 (green) and collagen IV (red). The bottom image on each panel shows a cross section prepared from the same retina (red: DAPI, green: CD31).

Figure 19 demonstrates that crystalline aA is regulated by an increase in cells of the rescued nuclear outer layer after treatment with Lin-HCS but not in contralateral eyes treated with control cells. Left panel; IgG control in rescued retina, central panel; aA crystalline in rescued retina, right panel; aA crystalline in un rescued retina.

Figure 20 includes tables of genes that are regulated by increase in murine retinas that have been treated with Lin-HCS. (A) Genes whose expression increases 3 times in mouse retinas treated with murine Lin-HCS. (B) Crystalline genes that are regulated by an increase in mouse retinas treated with murine Lin-HSC. (C) Genes whose expression increases twice in mouse retinas treated with human Lin-HCS. (D) Genes for neurotrophic factors or growth factors whose expression is regulated by an increase in mouse retinas treated with human Lin-HCS.

Figure 21 illustrates the distribution of surface antigens CD31 and integrin a6 in human Lin-HSC populations positive for CD133 (DC133 +) and negative for CD133 (CD133-). The left panels show flow cytometry scatter plots. Center and right panels are histograms that show the level of expression of specific antibodies in the cell population. The Y axis represents the number of events and the X axis shows the signal strength. A filled histogram shifted to the right of the histogram depicted in contour (control) represents an increase in fluorescent signal and antibody expression above the background level.

Figure 22 illustrates the postnatal retinal development in wild-type C57 / B16 mice raised in normal oxygen levels (normoxia), days after birth P0 through P30.

Figure 23 illustrates the model of oxygen-induced retinopathy in C57 / B16 mice raised at high oxygen levels (hyperoxia; 75% oxygen) between P7 and P12, followed by normoxia from P12-P17.

Figure 24 demonstrates vascular salvage by treatment with Lin-HSC populations in the oxygen-induced retinopathy (OIR) model.

Figure 25 shows that photoreceptors rescued in the outer nuclear layer (ONL) of rd1 mice after intravitreal injection of Lin-HSC are predominantly cones. A small percentage of photoreceptors in the retina of wild-type mice (upper panel) were cones as demonstrated by opsin expression of red / green cones (A) while the majority of ONL cells were positive for rhodopsin specific red (B). Autofluoresce retinal vasculature with preimmune serum (C) but the nuclear layers were completely negative for staining with specific opsins of cones or rods. Retinas of rd / rd mice (lower panels) had a decreased internal nuclear layer and an almost completely atrophic ONL, both of which were negative for opsin from cones (D) or rods (panel G). Eyes treated with HSC CD31-control were identical to non-injected rd / rd retinas, without any staining for opsin from cones (E) or rods (H). The contralateral eyes treated with Lin-HSC showed a markedly reduced ONL, although clearly present ONL that is predominantly composed of cones, as demonstrated by positive immunoreactivity for red / green cones opsin (F). A small number of canes (I) was also observed.

Figure 26 shows scatter plots from the characterization by flow cytometry of stem cell populations of negative lineage and positive lineage (upper left and lower left graphs, respectively) showing the percentages of cells expressing the CD44 antigen (points of data in red); as well as graphs of populations of negative cells for CD31 and positive for CD31 (upper right and lower right graphs, respectively), which show the percentages of cells expressing the CD44 antigen (data points in red).

Figure 27 shows scatter plots from the flow cytometry characterization of a population of negative lineage cells expressing a significant level of CD44 antigen (set of graphs on the left) and a subpopulation of bone marrow cells that they do not express a significant level of CD44 antigen (set of graphs on the right) that illustrate the relative percentages of cells expressing various other cell surface antigens.

Figure 28 shows microphotographic images of a retina of a mouse in which cells from the population of preferred isolated MLBM cells were injected intravitreally compared to a retina of a mouse in which cells were injected intravitreally CD44lo.

Figure 29 shows microphotographic images of eye retinas in which cells from the MLBM cell population (CD44hi) and CD44lo cells were injected.

Figure 30 shows bar graphs demonstrating the beneficial effects of the MLBM cell population to improve pathogenic angiogenesis and favor the beneficial physiological revascularization of mouse retinas in the oxygen-induced retinopathy model of prematurity retinopathy. The upper graph compares the area of preretinal neovascular glomeruli for control retina (first bar), retina treated with CD44lo cells (central bar) and retinas treated with cells from the MLBM cell population (right bar). The graph below compares the area of vascular obliteration for control retina (first bar), retina treated with CD44lo cells (central bar) and retinas treated with cells from the MLBM cell population (right bar).

Figure 31 is a microphotographic image demonstrating that once cells of the MLBM cell population have been incorporated into the retinal vasculature, the cells express vascular endothelial growth factor (VEGF), as indicated by green staining. of the cells at the bottom of the image.

Figure 32 depicts microphotographic images demonstrating that cells from the population of MLBM CD11b + cells selectively select the retinal vasculature.

Figure 33 depicts microphotographic images demonstrating that CD44-CD11b bone marrow cells selectively select the retinal vasculature as a target.

Figure 34 shows the amino acid residue sequence of the TrpRS T2 fragment (SEQ ID NO: 3) and the T2-TrpRSGD variation thereof (SEQ ID NO: 4).

Figure 35 shows the amino acid residue sequence of mini-TrpRS (SEQ ID NO: 5).

Figure 36 shows the amino acid residue sequence of T1-TrpRS (SEQ ID NO: 6).

Detailed description of the preferred embodiments

The bone marrow includes hematopoietic stem cells that can develop in various types of blood cells, for example, B cells, T cells, granulocytes, platelets and erythrocytes. Lineage surface antigens are a group of cell surface proteins that are markers of mature blood cell lineages, including CD2, CD3, CD11, CD11a, Mac-1 (CD11b: CD18), CD14, CD16, CD19, CD24 , CD33, CD36, CD38, CD45, CD45RA, murine Ly-6G, murine TER-119, CD56, CD64, CD68, CD86 (B7.2), CD66b, human leukocyte antigen DR (HLA-DR) and CD235a (glycoforin A ). Isolated hematopoietic stem cells that do not express significant levels of a "lineage surface antigen" (Lin) on their cell surfaces are referred to herein as "negative lineage" or "Lin-" hematopoietic stem cells, that is, Lin- HSC Human hematopoietic stem cells commonly express other surface antigens such as CD31, CD34, CD117 (c-kit) and / or CD133. Murine hematopoietic stem cells commonly express other surface antigens such as CD34, CD117 (c-kit), Thy-1 and / or Sca-1.

Bone marrow cells include a subpopulation of cells that express the CD44 antigen (i.e., the hyaluronic acid receptor) and CD11b (aM integrin). A population of myeloid-type bone marrow cells enriched in cells expressing CD44 and CD111b can be isolated from the bone marrow by treating the bone marrow cells with an antibody against the CD44 antigen (anti-CD44) and / or a antibody against the CD11b antigen (anti-CD11b), and then selecting the cells that immunoreact with the antibody. The antibody can then be separated from the cells by procedures that are well known in the art. Cells can be selected, for example, using flow cytometry, using antibodies bound to or coated on beads followed by filtration, or other separation procedures that are well known in the art. A majority of the selected cells are of negative lineage and express both the CD44 antigen and the CD11b antigen, regardless of which antibody is used in the isolation.

Stem cells are normally identified by the distribution of antigens on the surface of cells (for a detailed discussion see Stem Cells: Scientific Progress and Future Directions, a report prepared by the National Institutes of Health, Office of Science Policy, June 2001 , Appendix E: Stem Cell Markers, which is incorporated herein by reference in the relevant grade). Approximately 75% of hematopoietic stem cells of negative lineage isolated from the bone marrow are also positive for CD44. In a preferred embodiment, a majority of the cells in the MLBM cell population are hematopoietic stem cells of negative lineage (i.e., CD44 + Lin-HSC).

As used herein and in the appended claims, the term "adult" in reference to bone marrow and bone marrow cells includes bone marrow isolated after birth, that is, from juvenile and adult individuals, as opposed to embryos Therefore, the term "adult mammal" refers to juvenile (postnatal) and fully mature mammals, as opposed to an embryo or prenatal individual.

Lin-HSC populations containing endothelial progenitor cells (EPC) are particularly useful. Isolated Lin-HSC populations preferably comprise mammalian cells in which at least about 20% of the cells express the CD31 surface antigen, which is commonly present in endothelial cells. In another embodiment, at least about 50% of the cells express CD31, more preferably at least about 65%, most preferably at least about 75%. Preferably, at least about 50% of the cells of the Lin-HSC populations preferably express the integrin a6 antigen.

In a population of preferred murine Lin-HSC, at least about 50% of the cells express the CD31 antigen and at least about 50% of the cells express the CD117 antigen (c-kit). Preferably, at least about 75% of the Lin-HSC cells express the CD31 surface antigen, more preferably about 81% of the cells. In another preferred murine embodiment, at least about 65% of the cells express the CD117 surface antigen, more preferably about 70% of the cells. A particularly preferred embodiment is a Lin-HCS population in which from about 50% to about 85% of the cells express the CD31 surface antigen and from about 70% to about 75% of the cells express CD117 surface antigen.

In a preferred murine Lin-HSC population, the cells are negative for CD133, at least about 50% of the cells express the CD31 surface antigen, and at least about 50% of the cells express the integrin a6 antigen. Still another preferred embodiment is a population of human Lin-HSC in which the cells are positive for CD133, in which less than about 30% of the cells express the CD31 surface antigen and less than about 30% of the cells. cells express the integrin a6 antigen.

Isolated Lin-HSC populations select astrocytes selectively and are incorporated into the retinal neovasculature when injected intravitreally into the eye of mammalian species, such as a mouse or a human being, from which the cells were isolated.

Populations of isolated MLBM cells also selectively select astrocytes as target and are incorporated into the retinal neovasculature when injected intravitreally into the eye of mammalian species, such as a mouse or a human being, from which the cells were isolated.

Isolated MLBM cell populations include cells that differentiate into endothelial cells and generate vascular structures within the retina. In particular, populations of MLBM cells are useful for the treatment of degenerative vascular diseases of the retina and neovascular of the retina, and for the repair of vascular lesion of the retina. MLBM cell populations also promote neuronal rescue in the retina and promote regulation by increasing antiapoptotic genes. MLBM cell populations are particularly useful for treating retinal defects in the eyes of neonatal mammals, such as mammals suffering from oxygen-induced retinopathy or retinopathy of prematurity.

The number of cells injected into the eye is sufficient to stop the pathological state of the eye. For example, the amount of cells injected may be effective in repairing the lesion in the retina of the eye, stabilizing the neovasculature of the retina, maturing the neovasculature of the retina, and preventing or repairing vascular leakage and vascular bleeding.

Antiangiogenic (i.e., angiostatic) fragments of TrpRS are, for example, the T1 and T2 fragments of TrpRS, which are described in detail in the US patent application being processed together with the present, jointly owned, with serial number 10 /080,839. Transfected cells used according to the present invention are useful for the treatment of retinal diseases that involve abnormal vascular development, such as diabetic retinopathy, and similar diseases. Preferably, the cell populations are those of human cells.

Transfected cells may include a gene that operatively encodes a neurotrophic agent such as a nerve growth factor, neurotrophin-3, neurotrophin-4, neurotrophin-5, ciliary neurotrophic factor, neurotrophic factor derived from the pigmented epithelium of the retina, factor of insulin-like growth, neurotrophic factor derived from the glial cell line, neurotrophic factor derived from the brain and the like. Such neurotrophic cells are useful for promoting neuronal rescue in degenerative neuronal diseases of the retina such as glaucoma and retinitis pigmentosa, in the treatment of retinal nerve lesions and the like. It has been reported that ciliary neurotrophic factor implants are useful for the treatment of pigmentary retinitis (see Kirby et al. 2001, Mol Ther. 3 (2): 241-8; Farrar et al. 2002, EMBO Journal 21: 857-864 ). The brain-derived neurotrophic factor modulates genes reported to be associated with growth in injured retinal ganglia (see Fournier, et al., 1997, J. Neurosci. Res. 47: 561-572). The neurotrophic factor derived from the glial cell line delays as reportedly degenerating photoreceptors in pigmentary retinitis (see McGee et al. 2001, Mol Ther. 4 (6): 622-9).

Preferably, at least about 1 x 105 cells are administered by intravitreal injection to a mammalian eye suffering from a degenerative disease of the retina. The amount of cells to be injected may depend on the severity of the retinal degeneration, the age of the mammal and other factors that will be apparent to a person skilled in the art of treating retinal diseases. The cells can be administered in a single dose or by administration of multiple doses over a period of time, as determined by the doctor in charge of the treatment.

The results of the ROP OIR model indicate that a particular advantage is a vasculotrophic and neurotrophic rescue effect observed in eyes treated intravitreally with cells from hematopoietic stem cell populations of negative lineage. Neurons and photoreceptors of the retina, particularly the cones, are preserved and a certain amount of visual function can be preserved in eyes treated with cells of the hematopoietic stem cell populations of negative lineage. Treatment with angiogenesis inhibitors did not block the therapeutic effects of hematopoietic stem cell populations of negative lineage. Macrophage-like cells were observed in conjunction with the rescued blood vessels, suggesting a possible link between immune cells and bone marrow cells of negative lineage in the vasculotrophic activity of negative lineage cells. However, the injection of antibodies against CD18 between P7 and P17, to reduce macrophage extravasation from blood vessels did not block the rescue effects of hematopoietic stem cells of negative lineage.

The cells can be injected before the onset of the disease or in the early stages of the disease to protect the retina from injury that would otherwise result if the eye were left untreated. Such prophylactic treatment is particularly desirable in cases in which it is known that the mammal to be treated is at risk of developing retinopathy of prematurity, oxygen-induced retinopathy or other retinopathic diseases. Prophylactic treatments are particularly effective, since the presence of negative lineage cell populations in the eye really attenuates the severity of the vascular lesion in the retina, and can stop the disease before the injury occurs, as opposed to Simply favor damage recovery. For example, in the OIR model, mice that are injected with Lin-HSC between P3 and P7, before hyper-toxic exposure, suffer less retinal damage and recover faster than mice that are injected after hyperxic exposure. In addition, mice that were treated during hyperoxic exposure also demonstrated accelerated recovery.

Vascular development of the murine retina.

A model for ocular angiogenesis. The mouse eye provides a recognized model for the study of vascular development of the mammalian retina, such as the vascular development of the human retina. During the development of the murine retinal vasculature, retinal blood vessels produced by ischemia develop in close association with astrocytes. These glial elements migrate to the retina of the human fetus, or the neonatal rodent, in the third trimester from the optic disc along the ganglion cell layer and spread radially. As the murine retinal vasculature develops, endothelial cells use this established astrocytic template to determine the vascular pattern of the retina (see Figure 1 (a and b)). Figure 1 (a and b) represents schematic diagrams of the developing mouse retina. The panel (a) represents the development of the primary plexus (dark lines in the upper left part of the diagram) superimposed on the astrocyte mold (light lines) while (b) represents the second phase of retinal vessel formation. In Figure 1, GCL means the ganglion cell layer; IPL means the internal plexus layer; INL means the inner nuclear layer; OPL means the outer plexus layer; ONL means the outer nuclear layer; RPE means the retinal pigment epithelium; ON means the optic nerve; and P means the periphery.

At birth, the retinal vasculature is practically absent. By the 14th postnatal day (P14), the retina has developed the complex primary (superficial) and secondary (deep) layers of retinal vessels coinciding with the appearance of vision. Initially, ray-like peripapillary vessels grow radially along the pre-existing astrocytic network towards the periphery, progressively interconnecting by the formation of the capillary plexus. These vessels grow as a monolayer within the nerve fiber up to P10 (Figure 1 (a)). Between P7-P8, collateral branches start to sprout from this primary plexus and penetrate the retina into the outer plexiform layer in which they form the secondary, or deep, retinal plexus. Towards P21, the entire network undergoes extensive remodeling and a tertiary or intermediate plexus forms on the inner surface of the inner nuclear layer (Figure 1 (b)).

The neonatal mouse retinal angiogenesis model is useful for studying the role of HSC during ocular angiogenesis for several reasons. In this physiologically relevant model, there is a large astrocytic mold before the appearance of endogenous blood vessels, which allows an evaluation of the role of cell-cell targeting during a neovascular process. In addition, this vascular process of the constant and reproducible nenonatal retina is known to be caused by hypoxia, having in this sense similarities with many diseases of the retina in which it is known that ischemia plays a role.

Enrichment of endothelial progenitor cells (EPC) from the bone marrow.

Although the expression of cell surface markers in the EPC population found in HSC preparations has been extensively evaluated, markers that uniquely identify EPC are still poorly defined. To enrich EPC, hematopoietic (Lin +) lineage positive marker cells were reduced, that is, B lymphocytes (CD45), T lymphocytes (CD3), granulocytes (Ly-6G), monocytes (CD11) and erythrocytes (TER119), of cells Mononuclear bone marrow of mice. The Sca-1 antigen was used to further enrich the EPC antigen. A comparison of the results obtained after intravitreal injection of identical numbers of either Lin-Sca-1 + cells or Lin- cells detected differences between the two groups. In fact, when only Lin-Sca-1- cells were injected, a much greater incorporation was observed in developing blood vessels.

Lin-HSC populations are enriched with EPC, based on functional assays. In addition, Lin + HSC populations behave functionally quite differently from Lin-HSC populations. The epitopes commonly used to identify EPC for each fraction were also evaluated (based on previously reported in vitro characterization studies). Although none of these markers was exclusively associated with the Lin- fraction, they all increase from approximately 70 to approximately 1800% in the Lin-HSC, compared to the Lin + HSC fraction (Figure 1 (c)). Figure 1, panel (c) illustrates the flow cytometry characterization of separate Lin + HSC and Lin-HSC cells derived from bone marrow. The top row of panel (c) shows a dot plot distribution of hematopoietic stem cells from cells not labeled with antibody. R1 defines the quantifiable-activated area of positive PE staining; R2 indicates positive for GFP. The Lin-HSC dot plots are shown in the center row and the Lin + HSC dot plots are shown in the bottom row. C57B / 6 cells were labeled with antibodies conjugated to PE for Sca-1, c-kit, Flk-1 / KDR, CD31. Tie-2 data were obtained from Tie-2-GFP mice. The percentages in the corners of the dot plots indicate the percentage of positively marked cells of the total Lin-o Lin + HSC population. Interestingly, accepted EPC markers such as Flk-1 / KDR, Tie-2 and Sca-1 were poorly expressed and therefore not used for further fractionation.

Lin-HSC (a) can be isolated by extracting bone marrow from an adult mammal; (b) separating a plurality of monocytes from the bone marrow; (c) labeling monocytes with biotin-conjugated lineage panel antibodies against one or more lineage surface antigens, preferably lineage surface antigens selected from the group consisting of CD2, CD3, CD4, CD11, CD11a, Mac -1, CD 14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, Ly-6G (murine), TER-119 (murine), CD45RA, CD56, CD64, CD68, CD86 (B7.2), CD66b , human leukocyte antigen DR (HLA-DR) and CD235a (glycophorin A); (d) eliminating monocytes that are positive for said one or more lineage surface antigens from the plurality of monocytes; and (e) recovering a population of hematopoietic stem cells of negative lineage from it.

When the Lin-HSCs are isolated from adult human bone marrow, preferably the monocytes are labeled with biotin-conjugated lineage panel antibodies against lineage surface antigens CD2, CD3, CD4, CD11a, Mac-1, CD14, CD16, CD19, CD33, CD38, CD45RA, CD64, CD68, CD86 (B7.2) and CD235a. When the Lin-HSCs are isolated from adult murine bone marrow, monocytes are preferably labeled with biotin-conjugated lineage panel antibodies against CD3, CD11, CD45, Ly-6G and TER-119 lineage surface antigens.

The HSC Lin-injected intravitreal cells contain EPCs that select astrocytes as target and are incorporated into the vasculature of the developing retina.

To determine whether Lin-HSC injected intravitreally can select specific types of retinal cells as target, use the astrocytic template and participate in retinal angiogenesis, approximately 105 cells of a Lin-HSC composition or Lin + cells were injected HSC (control, approximately 105 cells) isolated from the bone marrow of adult mice (transgenic for GFP or LacZ) in mouse eyes on postnatal day 2 (P2). Four days after injection (P6), many cells of the Lin-HSC composition, derived from transgenic mice for GFP or LacZ were adherent to the retina and had the characteristic elongated appearance of endothelial cells (Figure 2 (a)). Figure 2 illustrates the grafting of Lin- cells in the developing mouse retina. As shown in Figure 2, panel (a), the eGFP + Lin-HSC injected intravitreally four days after injection (P6) bind and differentiate into the retina.

In many regions of the retinas, GFP-expressing cells were arranged in a pattern that adapted to the underlying astrocytes and resembled blood vessels. These fluorescent cells were observed ahead of the developing endogenous vascular network (Figure 2 (b)). Conversely, only a small number of Lin + HSC (Figure 2 (c)), or adult mouse mesenteric endothelial cells (Figure 2 (d)) were attached to the surface of the retina. In order to determine if cells from a population of injected Lin-HSC could also bind retinas with established vessels, a Lin-HSC composition was injected into adult eyes. Interestingly, it was not observed that cells were bound to the retina or incorporated into established, normal retinal blood vessels (Figure 2 (e)). This indicates that Lin-HSC compositions do not alter a normally developed vasculature and will not initiate abnormal vascularization in normally developed retinas.

In order to determine the relationship between injected Lin-HSC compositions and retinal astrocytes, a transgenic mouse was used, expressing glial fibrillary acidic protein (GFAP, an astrocyte marker) and promoter-directed green fluorescent protein (GFP). Examination of the retinas of these GFAP-GFP transgenic mice that were injected Lin-HSC of transgenic mice for eGFP demonstrated the joint location of the injected EPC eGFPs and the existing astrocytes (Figure 2 (f-h), arrows). The eGFP + Lin-HSC processes were observed to adapt to the underlying astrocytic network (arrows, figure 2 (g)). Examination of these eyes showed that injected, labeled cells only bound to astrocytes; in P6 mouse retinas, in which the periphery of the retina does not yet have endogenous vessels, injected cells adherent to astrocytes were observed in these areas not yet vascularized. Surprisingly, marked cells were observed in the deepest layers of the retina at the precise location where retinal vessels will develop later (Figure 2 (i), arrows).

To determine if injected Lin-HSCs are stably incorporated into the vasculature of the developing retina, retinal vessels were examined at several subsequent time points. As soon as P9 (seven days after injection), Lin-HSC were incorporated into CD31 + structures (Figure 2 (j)). By P16 (14 days after injection), the cells were already extensively incorporated into vascular-like structures of the retina (Figure 2 (k)). When rhodamine-dextran was injected intravascularly (to identify functional retinal blood vessels) before sacrificing the animals, most Lin-HSCs aligned with patent vessels (Figure 2 (1)). Two patterns of labeled cell distribution were observed: (1) in one pattern, the cells were interspersed along the vessels between unlabeled endothelial cells; and (2) the other pattern showed that the vessels were composed entirely of labeled cells. Cells injected into vessels of the deep vascular plexus were also incorporated (Figure 2 (m)). Although sporadic incorporation of EPC derived from Lin-HSC into the neovasculature has been reported previously, this is the first report of vascular networks composed entirely of these cells. This demonstrates that cells from a population of Lin-HSC derived from the bone marrow, injected intravitreally, can be effectively incorporated into any layer of the vascular plexus of the retina in formation.

Histological examination of non-retinal tissues (for example, brain, liver, heart, lung, bone marrow) did not demonstrate the presence of any GFP positive cells when examined up to 5 or 10 days after intravitreal injection. This indicates that a subpopulation of cells within the Lin-HSC fraction selectively targets retinal astrocytes and is stably incorporated into the vasculature of the developing retina. Since these cells have many characteristics of endothelial cells (association with retinal astrocytes, elongated morphology, stable incorporation into patent vessels and not present in extravascular locations), these cells represent EPCs present in the Lin-HSC population. Astrocytes selected as target are the same type observed in many of the hypoxic retinopathies. It is well known that glial cells are a prominent component of the neovascular fronds of glomeruli observed in DR and other forms of retinal injury. Under conditions of reactive gliosis and neovascularization induced by ischemia, activated astrocytes proliferate, produce cytokines and regulate by GFAP increase, similar to what was observed during the formation of the nenonatal retinal vascular mold in many species of mammals including humans.

Lin-HSC populations will select astrocytes activated in adult mouse eyes as they do in neonatal eyes. Lin-HSC cells were injected into adult eyes with retinas injured by photocoagulation (Figure 3 (a)) or needle tip (Figure 3 (b)). In both models, a population of cells with prominent GFAP staining was observed only around the site of the lesion (Figure 3 (a and b)). Injected Lin-HSC composition cells were located at the site of the lesion and remained specifically associated with astrocytes positive for GFAP (Figure 3 (a and b)). At these sites, it was also observed that Lin-HSC cells migrated to the deepest layer of the retina at a level similar to that observed during the neonatal formation of the deep retinal vasculature. The uninjured portions of the retina did not contain Lin-HSC cells, identical to what was observed when Lin-HSC was injected into normal, uninjured adult retinas (Figure 2 (e)). These data indicate that Lin-HSC compositions can selectively select as activated glial cells in adult retinas injured with gliosis as well as neonatal retinas undergoing vascularization.

Lin-HSC injected intravitreally can rescue and stabilize the degenerating vasculature.

Since Lin-HSC compositions injected intravitreally select astrocytes as target and are incorporated into the normal retinal vasculature, these cells stabilize the degenerating vasculature in ischemic or degenerative retinal diseases associated with gliosis and vascular degeneration. The rd / rd mouse is a model for the degeneration of the retina that shows a deep degeneration of the vascular layers of the retina and photoreceptors one month after birth. The retinal vasculature in these mice develops normally until P 16, at which point the deepest vascular plexus experiences regression; In most mice, the deep and intermediate plexus have degenerated almost completely towards P30.

To determine if HSC can rescue the regression vessels, Lin + or Lin-HSC (from Balb / c mice) were injected into rd / rd mice intravitreally in P6. By P33, after injection with Lin + cells, the vessels of the deepest retinal layer were almost completely absent (Figure 4 (a and b)). In contrast, most retinas injected with Lin-HSC into P33 had an almost normal retinal vasculature with three well-formed parallel vascular layers (Figure 4 (a and d)). The quantification of this effect showed that the average length of the vessels in the deep vascular plexus of eyes of rd / rd to those who were injected Lin- was almost three times greater than in the eyes treated with untreated Lin + cells (Figure 4 (e)). Surprisingly, the injection of a Lin-HSC composition derived from the adult mouse bone marrow rd / rd (FVB / N) also rescued the vasculature of the degenerative rd / rd neonatal mouse retina (Figure 4 (f) ). The degeneration of the vasculature in mouse eyes rd / rd is observed already at 2-3 weeks after birth. Injection of Lin-HSC as late as P15 also resulted in partial stabilization of the degenerating vasculature in the rd / rd mice for at least one month (Figure 4 (g and h)).

A composition of Lin-HSC injected into younger rd / rd mice (eg, P2) was also incorporated into the developing superficial vasculature. Towards P11, it was observed that these cells migrate to the level of the deep vascular plexus and form an identical pattern to that observed in the vascular layer of the wild-type external retina (Figure 5 (a)). In order to describe more clearly the way in which cells of injected Lin-HSC compositions are incorporated into and stabilize the degenerating retinal vasculature in rd / rd mice, a Lin-HSC composition of mice was injected Balb / c in mouse eyes Tie-2-GFP FVB. FVB mice have the rd / rd genotype and because they express the Tie-2-GFP fusion protein, all endogenous blood vessels are fluorescent.

When unlabeled cells of a Lin-HSC composition are injected into neonatal Tie-2-GFP FVB eyes and subsequently incorporated into the developing vasculature, there should be unlabeled gaps in the vessels labeled with Tie-2-GFP, endogenous that correspond to the incorporated, unlabeled Lin-HSCs that were injected. Subsequently, staining with another vascular marker (for example, CD-31) then delineates the entire vessel, allowing the determination of whether non-endogenous endothelial cells are part of the vasculature. Two months after injection, CD31-positive vessels, negative for Tie-2-GFP were observed in the eye retinas in which the Lin-HSC composition was injected (Figure 5 (b)). Interestingly, most of the rescued vessels contained Tie-2-GFP positive cells (Figure 5 (c)). The distribution of pericytes, as determined by staining the smooth muscle actin, did not change by injection of Lin-HSC, independently

5 of whether there was vascular rescue (Figure 5 (d)). These data clearly demonstrate that Lin-HSC cells injected intravitreally migrate to the retina, participate in the formation of normal retinal blood vessels and stabilize the vasculature in endogenous degeneration in a genetically defective mouse.

Inhibition of retinal angiogenesis by transfected Lin-HSC cells.

Most vascular diseases of the retina involve abnormal vascular proliferation instead of degeneration. Astrocyte-directed transgenic cells can be used to deliver an antiangiogenic protein and inhibit angiogenesis. Cells of Lin-HSC compositions were transfected with T2-tryptophanyl-tRNA synthetase (T2-TrpRS). T2-TrpRS is a 43 kD fragment of TrpRS that potently inhibits the

15 retinal angiogenesis (figure 6 (a) and figure 34). In P12, the retinas of eyes that were injected with a composition of Lin-HSC transfected with plasmid (without T2-TrpRS gene) in P2 had primary retinal vascular plexuses (Figure 6 (c)) and secondary (Figure 6 ( d)) normal. When the composition of Lin-HSC transfected with T2-TrpRS was injected into P2 eyes and evaluated 10 days later, the primary network had significant abnormalities (Figure 6 (e)) and the formation of the deep retinal vasculature was almost inhibited. completely (figure 6 (f)). The few vessels observed in these eyes were markedly attenuated with large gaps between the vessels. The degree of inhibition by Lin-HCS secreting T2-TrpRS is detailed in Table 1.

T2-TrpRS is produced and secreted by cells in the Lin-HSC composition in vitro and after the injection of these transfected cells into the vitreous humor, a 30 kD fragment of T2-TrpRS was observed in the retina (Figure 6 (b )). This

25 kD fragment was specifically observed only in retinas in which transfected Lin-HSC were injected and this decrease in apparent molecular weight compared to recombinant or synthesized protein in vitro may be due to processing or degradation of T2-TrpRS in alive. These data indicate that Lin-HSC compositions can be used to deliver functionally active genes, such as genes expressing angiostatic molecules, to the retinal vasculature by targeting activated astrocytes. Although it is possible that the observed angiostatic effect is due to cell-mediated activity, this is very unlikely since the eyes treated with identical Lin-HSC compositions, but not transfected with T2 had a normal retinal vasculature.

Table 1. Vascular inhibition by Lin-HSC secreting T2-TrpRS 35

Primary plexus

Deep plexus

Inhibited

Normal Complete Partial Normal

T2-TrpRS

60% 40% 33.3% 60% 6.7%

(15 eyes)

(9 eyes) (6 eyes) (5 eyes) (9 eyes) (1 eye)

Control

 0% 100% 0% 38.5% 61.5%

(13 eyes)

(0 eyes)  (13 eyes) (0 eyes) (5 eyes) (8 eyes)

The populations of Lin-HSC injected intravitreally are located in astrocytes of the retina, are incorporated into vessels and can be useful in the treatment of many diseases of the retina. Although most of the injected HSC compositions adhere to the astrocytic mold, small numbers migrate deeply to the retina, targeting regions where the deep vascular network will develop later. Even if no positive astrocytes are observed for GFAP in this area before 42 days after birth, this does not rule out the possibility that GFAP negative glial cells are already present to provide a signal for the location of Lin-HSC. Previous studies have shown that many diseases are associated with reactive gliosis. In DR, in particular, glial cells and their extracellular matrix are associated with angiogenesis

45 pathological.

Since cells of the injected Lin-HSC compositions specifically bind to glial cells expressing GFAP, regardless of the type of lesion, Lin-HSC compositions can be used to target preangiogenic retinal lesions. For example, in ischemic retinopathies, such as diabetes, neovascularization is a response to hypoxia. By directing Lin-HSC compositions to pathological neovascularization sites, developing neovasculature can be stabilized by preventing neovasculature abnormalities such as hemorrhage or edema (the causes of vision loss associated with DR) and hypoxia that originally stimulated neovascularization can possibly be relieved. . Abnormal blood vessels can be restored to a normal state. In addition, angiostatic proteins, such as T2-TrpRS (SEQ ID NO: 3 in 55 Figure 34), T2-TrpRS-GD (SEQ ID NO: 4 in Figure 34), mini-TrpRS (SEQ ID NO: 5 in Figure 35) and T1-TrpRS (SEQ ID NO: 6 in Figure 36) can be delivered to pathological angiogenesis sites using transfected Lin-HSC compositions and laser-induced astrocyte activation. Preferred angiostatic fragments of TrpRS include T2-TrpRS and T2-TrpRS-GD. Since laser photocoagulation is commonly used in clinical ophthalmology, this approach has application for many diseases of the retina. Although such cell-based approaches have been investigated in cancer therapy, their use for diseases

Eyepieces are more advantageous since intraocular injection makes it possible to deliver large numbers of cells directly to the disease site.

Neurotrophic and vasculotrophic rescue by Lin-HSC.

MACS was used to separate Lin-HSC from the bone marrow of mice with enhanced green fluorescent protein (eGFP), C3H (rd / rd), FVB (rd / rd) as described above. Lin-HSC containing EPC of these mice were injected intravitreally in P6 C3H or FVB mouse eyes. Retinas were collected at various time points (1 month, 2 months and 6 months) after injection. Vasculature was analyzed by scanning with a confocal laser microscope after staining with antibodies against CD31 and retinal histology after nuclear staining with DAPI. Analysis of the gene expression of retinal mRNA microalignments at variable time points was also used to identify genes possibly involved in the effect.

The eyes of rd / rd mice had a profound degeneration of both the neurosensory retina and the vasculature of the retina towards P21. The eyes of rd / rd mice treated with Lin-HSC in P6 maintained a normal retinal vasculature for up to 6 months; both deep and intermediate layers improved significantly compared to controls at all time points (1M, 2M and 6M) (see Figure 12). In addition, it was observed that the retinas treated with Lin-HSC were also thicker (1M; 1.2 times, 2M; 1.3 times, 6M; 1.4 times) and had higher numbers of cells in the outer nuclear layer ( 1M; 2.2 times, 2M; 3.7 times, 6M; 5.7 times) in relation to eyes treated with Lin + HSC as a control. Large-scale genomic analysis of “rescued” rd / rd retinas (eg, Lin-HSC) compared to control retinas (untreated or untreated with Lin-) demonstrated a regulation by significant increase in genes encoding sHSP (small heat shock proteins) and specific growth factors that correlated with vascular and neural rescue, including genes that code for the proteins listed in Figure 20, panels A and B.

Lin-HSC populations derived from the bone marrow significantly and reproducibly induced the maintenance of a normal vasculature and dramatically increased the layers of photoreceptors and other neuronal cells in the rd / rd mouse. This neurotrophic rescue effect correlated with the regulation by significant increase of small heat shock proteins and growth factors and provides an understanding of the therapeutic approaches for degenerative disorders of the retina currently untreatable.

Mouse rd1 / rd1 retinas have deep vascular and neuronal degeneration.

The vascular and neuronal development of the normal postnatal retina in mice has been well described and is analogous to changes observed in the human fetus in the third trimester (Dorrell et al., 2002, Invest. Ophthalmol. Vis. Sci. 43: 3500- 3510). Homozygous mice for the rd1 gene share many features of human retinal degeneration (Frasson et al., 1999, Nat. Med. 5: 1183-1187) and show rapid loss of photoreceptors (PR) accompanied by severe vascular atrophy such as result of a mutation in the gene that codes for PR cGMP phosphodiesterase (Bowes et al. 1990, Nature 347: 677-680). To examine the vasculature during retinal development and its subsequent degeneration, antibodies against collagen IV (CIV), an extracellular matrix protein (ECM) of the mature vasculature, and CD31 (PECAM-1), a marker for endothelial cells (figure 15). The rd1 / rd1 (C3H / HeJ) retinas developed normally until about the postnatal day (P) 8 when degeneration of the outer nuclear layer (ONL) containing photoreceptors began. The ONL degenerated rapidly and the cells died by apoptosis so that only a single layer of nuclei remained towards P20. Double staining of complete microscopic retinal preparations with antibodies against both CIV and CD31 revealed details of vascular degeneration in rd1 / rd1 mice similar to that described by others (Blanks et al., 1986, J Comp. Neurol. 254: 543-553). The vascular layers of the primary and deep retina seemed to develop normally up to P12, after which there is a rapid loss of endothelial cells as demonstrated by the absence of CD31 staining. Endothelial cells positive for CD31 were present in a normal distribution to P12 but quickly disappeared afterwards. Interestingly, positive staining for IVC was still present at all time points examined, suggesting that the vessels and the associated NDE formed normally, but only the matrix remained after P 13, at which time they were not observed. CD31 positive cells (Figure 15, central panels). The intermediate vascular plexus also degenerates after P21, but the progression is slower than that observed in the deep plexus (Figure 15, upper panel). The layers of vascular and neural cells of the retina of a normal mouse are shown for comparison with the mouse rd1 / rd1 (right panels, figure 15).

Neuroprotective effect of Lin-HCS derived from bone marrow in rd1 / rd1 mice.

Lin-HCS injected intravitreally are incorporated into the vasculature of the endogenous retina in the three vascular plexuses and prevent the degeneration of the vessels. Interestingly, injected cells were practically never observed in the outer nuclear layer. These cells are either incorporated into the retinal vessels in formation or are observed in close proximity to these vessels. Murine Lin-HCS (from C3H / HeJ) were injected intravitreally into C3H / HeJ mouse eyes (rd1 / rd1) in P6, together before the onset of degeneration. By P30, the eyes that were injected with control cells (CD31-) had the typical rd1 / rd1 phenotype, that is, almost complete degeneration of the deep vascular plexus and ONL was observed in each retina examined. The eyes injected with Lin-HCS maintained intermediate and deep vascular plexus of normal appearance. Surprisingly, significantly more cells were seen in the internuclear layer (INL) and ONL of eyes that were injected Lin-HSC than in eyes that were injected with control cells (Figure 16 (A)). This rescue effect of Lin-HCS could be observed at 2 months (Figure 16 (B)) and for up to 6 months after injection (Figure 16 (C)). The differences in vasculature of the intermediate and deep plexus of the eyes that were injected with Lin-HSC, as well as the INL and ONL that contained neuronal cells, were significant at all time points measured when rescued eyes were compared and not rescued (figure 16 (B and C)). This effect was quantified by measuring the total length of the vasculature (Figure 16 (D)) and counting the number of DAPI positive cell nuclei observed in the ONL (Figure 16 (E)). Simple linear regression analysis was applied to the data of all time points.

A statistically significant correlation was observed between vascular rescue and neuronal rescue (for example, thickness of ONL) in P30 (p <0.024) and P60 (p <0.034) in the eyes injected with Lin-HSC (Figure 16 (F)). The correlation remained high, although not statistically significant (p <0.14) at P 180 compared to retinas that were injected Lin-HSC with respect to retinas that were injected with control cells (Figure 16 (F )). In contrast, the retinas in which control cells were injected showed no significant correlation between vasculature preservation and ONL at any time point (Figure 16 (F)). These data demonstrate that intravitreal injection of Lin-HCS results in vascular and neuronal rescue of the concomitant retina in retinas of rd1 / rd1 mice. No cells injected into the ONL or any place other than within, or in close proximity to, the retinal blood vessels were observed.

Functional rescue of rd / rd retinas that were injected Lin-HSC.

Electroretinograms (ERG) were performed in mice 2 months after injection of murine control cells or Lin-HCS (Figure 17). Immunohistochemical and microscopic analysis was performed with each eye after ERG records to confirm that vascular and neuronal rescue had occurred. The ERG records representative of treated, rescued and control eyes, not rescued show that, in rescued eyes, the digitally subtracted signal (treated eyes less untreated) produced a clearly detectable signal with an amplitude of the order of 8-10 microvolts ( figure 17). Clearly, the signals of both eyes are severely anomalous. However, constant and detectable ERGs could be recorded from eyes treated with Lin-HSC. In all cases, the ERG of the control eye could not be detected. Although the amplitudes of the signals in the rescued eyes were considerably lower than normal, the signals were consistently observed whenever there was histological rescue and were in the order of magnitude of those reported by other gene-based rescue studies. Overall, these results are demonstrative of some degree of functional rescue in eyes treated with Lin-HCS.

The types of retinal rd / rd cells are predominantly cones.

Rescued and un rescued retinas were analyzed by immunohistochemistry with antibodies specific for cone or rod opsin. The same eyes used for the ERG records presented in Figure 17 were analyzed to detect the opsin of cones or rods. In wild-type mouse retinas, less than about 5% of the photoreceptors present are cones (Soucy et al. 1998, Neuron 21: 481-493) and immunohistochemical staining patterns observed with red / green cones opsin such as shown in figure 25 (A) or red rhodopsin as shown in figure 25 (B) agreed with this percentage of cone cells. When wild-type retinas were stained with preimmune IgG, no staining was observed in the neurosensory retinas other than autofluorescence of the blood vessels (Figure 25 (C)). Two months after birth, the retinas of non-injected rd / rd mice had an essentially atrophic outer nuclear layer that showed no staining with antibodies against opsin from red / green cones (Figure 25 (D)) or Rhodopsin (Figure 25 ( G)). The eyes injected with CD31-HSC control were not stained positively for the presence of opsin or cones (Figure 25 (E))) or sticks (Figure 25 (H)). In contrast, the contralateral eyes that were injected with Lin-HSC had about 3 to about 8 rows of nuclei in a conserved outer nuclear layer; Most of these cells were positive for cones opsin (Figure 25 (F)) with approximately 1-3% positive for red opsin (Figure 25 (I)). Notably, this is almost the inverse of what is usually observed in the normal mouse retina, which is dominated by rods. These data demonstrate that the injection of Lin-HSC conserves the cones for prolonged periods of time during which they would usually degenerate.

Lin-HCS derived from the human bone marrow (hBM) also rescue degenerating retinas.

Lin-HCS isolated from the human bone marrow behave similarly to murine Lin-HCS. Bone marrow was collected from human donors and the Lin + HCS were reduced, producing a population of human Lin-HCS (hLin-HCS). These cells were labeled ex vivo with fluorescent dye and injected into C3SnSmn.CB17-Prkdc SCID mouse eyes. Injected hLin-HCS migrated to, and selected as target, retinal angiogenesis sites in an identical manner to that observed when murine Lin-HCS were injected (Figure 18 (A)). In addition to vascular targeting, human Lin-HCS also provided a robust rescue effect on both the vascular and neuronal cell layers of the rd1 / rd1 mice (Figure 18 (B and C)). This observation confirms the presence of cells in human bone marrow that select the retinal vasculature as target and can prevent retinal degeneration.

Lin-HCS have vasculotrophic and neurotrophic effects in the mouse rd10 / rd10.

Although the mouse rd1 / rd1 is the most widely used and best characterized model for retinal degeneration (Chang et al. 2002, Vision Res. 42: 517-525), degeneration is very rapid and in this sense differs from the course of usual, slower time observed in human disease. In this variety, photoreceptor cell degeneration begins approximately at P8, a time when the retinal vasculature is still rapidly expanding (Figure 15). The subsequent degeneration of the deep retinal vasculature occurs even while the intermediate plexus is still forming and, therefore, the retinas of rd1 / rd1 mice never fully develop, unlike what is observed in most humans With this disease. A rd10 mouse model was used, which has a slower time course of degeneration and more closely resembles the degenerative state of the human retina, to investigate the Lin-HSC-mediated vascular rescue. In the rd10 mouse, the degeneration of photoreceptor cells begins approximately in P21 and vascular degeneration begins shortly thereafter.

Since the development of the normal sensorineural retina is largely completed by P21, it is observed that degeneration begins after the differentiation of the retina has been completed and thus is more analogous to the degenerations of the human retina than the mouse model rd1 / rd1. Lin-HCS or control cells of rd10 mice were injected into P6 eyes and retinas were evaluated at varying time points. At P21, the eye retinas that were injected with both Lin-HSC and control cells appeared normal with complete development of all vascular layers and normal development of the INL and ONL (Figure 18 (D and H)). At approximately P21, degeneration of the retina began and progressed with age. By P30, the retinas in which control cells were injected had severe vascular and neuronal degeneration (Figure 18 (I)), while the retinas injected with Lin-HSC maintained almost normal photoreceptor cells and vascular layers ( Figure 18 (E)). The difference between rescued and un rescued eyes was more pronounced at later time points (compare Figure 18 (F and G) with 18 (J and K)). In the control treated eyes, the progression of vascular degeneration was more clearly observed by immunohistochemical staining for CD31 and collagen IV (Figure 18 (I-K)). Control treated eyes were almost completely negative for CD31, while positive vascular "clues" for collagen IV remained evident, indicating that vascular regression had occurred, rather than incomplete vascular formation. In contrast, eyes treated with Lin-HSC had positive vessels for both CD31 and collagen IV that looked very similar to normal, natural-type eyes (see Figure 18 (F and I)).

Gene expression analysis of mouse rd / rd retinas after treatment with Lin-HSC.

A large-scale genomic analysis (micro-alignment analysis) was used to analyze rescued and unrescued retinas to identify potential neurotrophic rescue mediators. Gene expression was compared in rd1 / rd1 mouse retinas treated with Lin-HCS with non-injected retinas as well as with retinas in which control cells were injected (CD31-). Each of these comparisons was made in triplicate. In order to be considered present, the genes were required to have an expression level at least 2 times higher than the background levels in the three triplicates. Genes were compared that were regulated by 3-fold increase in retinas protected with Lin-HSC compared to rd / rd mouse retinas with injected control cells and without injected cells as shown in Figure 20, panels A and B. They were calculated the coefficient of variance (VOC) levels for the genes expressed by dividing the standard deviation by the average expression level of each cRNA repeat. In addition, the correlation between expression levels and noise variance was calculated by correlating the mean and standard deviation (SD). A correlation between the level of gene expression and the standard deviation for each gene was obtained, which allowed reliable thresholds of level of expression and background levels to be determined. Overall, the data was completely within acceptable limits (Tu et al. 2002, Proc. Natl. Acad. Sci. U S A 99: 14031-14036). The genes that are treated individually, then showed expression levels above these critical expression levels. "T test" values were also determined for paired data for the treated genes. In each case, the p values are reasonable (near or below 0.05), which shows that there are similarities between repetitions and probable significant differences between the different test groups. Many of the genes regulated by increase significantly, including MAD and Ying Yang-1 (YY-1) (Austen et al. 1997, Curr. Top. Microbiol. Immunol. 224: 123130.), code for proteins with functions that they involve the protection of cells against apoptosis. Several crystalline genes, which have sequence homology and functions similar to known thermal shock proteins that are involved in the protection of cells against stress, were also regulated by increase by treatment with Lin-HSC. The expression of a-crystalline was localized in the ONL by immunohistochemical analysis (Figure 19). Figure 19 shows that crystalline aA is regulated by an increase in the cells of the outer nuclear layer rescued after treatment with Lin-HCS but not in the contralateral eyes treated with control cells. The left panel shows staining with IgG (control) in the rescued retina. The middle panel shows crystalline AA in a rescued retina. The right panel shows aA crystalline in un rescued retina.

Messenger RNA from rd1 / rd1 mouse retinas rescued with human Lin-HCS was hybridized with human-specific Affymetrix U133A microalignment chips. After the rigorous analysis, several genes were found whose mRNA expression was specific to human being, above the background level, and significantly higher in retinas rescued with human Lin-HSC compared to retinas rescued with murine Lin-HSC and un rescued retinas in which human control cells were injected (Figure 20, panel C). CD6, a cell adhesion molecule expressed on the surface of primitive and newly differentiated CD34 + hematopoietic stem cells, and interferon alfa 13, another gene expressed by hematopoietic stem cells, were both found using the bioinformatic microalignment technique, validating the evaluation protocol. In addition, several growth factors and neurotrophic factors were expressed above the background by mouse retinal samples rescued with human Lin-HSC (Figure 20, panel D).

Markers for hematopoietic cells of compromised lineage were used to negatively select a population of Lin-HSC derived from bone marrow containing EPC. Although the subpopulation of Lin-HSC derived from bone marrow that can serve as EPC is not characterized by commonly used cell surface markers, the behavior of these cells in the vasculature of the developing or injured retina is completely different from that observed for populations of adult endothelial cells or Lin +. These cells selectively select retinal angiogenesis sites and participate in the formation of patent blood vessels.

Inherited degenerative retinal diseases are often accompanied by loss of retinal vasculature. The effective treatment of such diseases requires the restoration of function, as well as the maintenance of complex tissue architecture. Although several recent studies have investigated the use of cell-based delivery of trophic factors or of the stem cells themselves, some combination of both may be necessary. For example, the use of growth factor therapy to treat degenerative retinal disease resulted in an unregulated excess growth of blood vessels resulting in severe alteration of the architecture of normal retinal tissue. The use of stem, retinal or neural cells to treat degenerative retinal disease can reconstitute neuronal function, but a functional vasculature will also be necessary to maintain the functional integrity of the retina. The incorporation of cells from a Lin-HSC population in the retinal vessels of rd / rd mice stabilized the degenerative vasculature without altering the structure of the retina. This rescue effect was also observed when cells were injected into rd / rd P15 mice. Since vascular degeneration begins at P16 in rd / rd mice, this observation widens the therapeutic window for treatment with effective Lin-HSC. Neurons and retinal photoreceptors are preserved and visual function is maintained in the eyes in which Lin-HSC cells are injected.

Lin-HCS derived from the adult bone marrow exert profound vasculotrophic and neurotrophic effects when injected intravitreally in mice with degenerative retinal disease. This rescue effect persists for up to 6 months after treatment and is most effective when Lin-HCS is injected before degeneration of the entire retina (up to 16 days after birth in mice that normally show degeneration of the entire retina towards 30 days after birth). This rescue is observed in two mouse models of retinal degeneration and, notably, can be carried out with HCS derived from the bone marrow of an adult human being when the recipient is an immunodeficient rodent with degeneration of the retina (for example, the SCID mouse) or when the donor is a mouse with degeneration of the retina. Although several recent reports have described a partial phenotypic rescue in mice or dogs with retinal degeneration after virus-based gene rescue with the wild-type gene (Ali, et al. 2000, Nat Genet 25: 306-310; Takahashi et al 1999, J. Virol. 73: 78127816; Acland et al. 2001, Nat. Genet. 28: 92-95.), The present invention is the first generic cell-based therapeutic effect achieved by vascular rescue. Therefore, the possible utility of such an approach in the treatment of a group of diseases (eg, pigmentary retinitis) with more than 100 known associated mutations is more practical than creating individual gene therapies to treat each known mutation.

The precise molecular basis of the neutrophic rescue effect remains unknown, but is observed only when there is concomitant vascular stabilization / rescue. The presence of injected stem cells, per se, is not sufficient to generate a neurotrophic rescue and the clear absence of neurons derived from stem cells in the outer nuclear layer rules out the possibility that the injected cells are becoming photoreceptors. The data obtained through microalignment analysis of gene expression demonstrated a regulation by significant increase in genes that are known to have antiapoptotic effects. Since most of the neuronal death observed in retinal degenerations occurs through apoptosis, such protection can be of great therapeutic benefit to prolong the life of photoreceptors and other critical neurons for visual function in these diseases. C-myc is a transcription factor that participates in apoptosis by regulating by increasing various factors of induction of apoptosis in the sense of 3 ’. C-myc expression increased 4.5 times in rd / rd mice with respect to the natural type, indicating the possible involvement in photoreceptor degeneration observed in the rd1 / rd1 mouse. It is known that Mad1 and YY-1, two genes regulated by drastically increased retinas protected with Lin-HSC (Figure 20, panel A), suppress c-myc activity, thereby inhibiting c-myc-induced apoptosis. It has also been shown that Mad1 overexpression suppresses Fas-induced activation of caspase-8, another critical component of the apoptotic pathway. The regulation by increase of these two molecules can play a role in the protection of the retina against vascular and neural degeneration, preventing the onset of apoptosis that normally leads to degeneration in rd / rd mice.

Another set of genes that were regulated by greatly increased retinas protected with Lin-HSC includes members of the crystalline family (Figure 20, panel B). Similar to thermal shock proteins and other stress-induced proteins, crystalline proteins can be activated by retinal stress and provide a protective effect against apoptosis. Abnormally low expression of crystalline aA correlates with loss of photoreceptors in a rat model of retinal dystrophy and a recent proteomic analysis of the retinal mouse in the rd / rd mouse demonstrated the induction of regulation by increased crystalline protein in response to Degeneration of the retina Based on the microalignment data of mouse rd / rd retinas rescued with EPC, regulation by increased crystalline proteins seems to play a key role in the neuroprotection of the EPC-mediated retina.

Genes such as c-myc, Mad1, Yx-1 and the crystalline are likely to be mediators in the 3 ’sense of neuronal rescue. Neurotrophic agents can regulate antiapoptotic gene expression, although microalignment analyzes of retinas rescued with mouse stem cells did not demonstrate induction of increased levels of known neurotrophic factors. Analysis of the rescue mediated by stem cells derived from human bone marrow with specific human chips showed, on the other hand, low but significant increases in the expression of multiple growth factor genes.

Incrementally regulated genes include several members of the fibroblast and otoferlin growth factor family. Mutations in the otoferlin gene are associated with genetic disorders that lead to deafness due to auditory neuropathy. It is possible that the production of otoferlin by injected Lin-HCS also contributes to the prevention of retinal neuropathy. Historically, it has long been assumed that the vascular changes observed in patients and animals with retinal degeneration were secondary to the decrease in metabolic demand as the photoreceptors die. The present data indicate that, at least for mice with inherited retinal degeneration, preserving the normal vasculature can also help maintain the components of the outer nuclear layer. Recent reports in the literature would support the concept that tissue-specific vasculature has trophic effects that go beyond those expected to simply provide vascular "nutrition." For example, liver endothelial cells can be induced to produce, after activation of VEGFR1, critical growth factors for the regeneration and maintenance of hepatocytes in the face of liver injury (LeCouter et al. 2003, Science 299: 890- 893).

Similar indicative interactions between vascular endothelial cells and adjacent hepatic parenchymal cells are supposedly involved in the organogenesis of the liver, long before the formation of functional blood vessels. Endogenous retinal vasculature in individuals with retinal degeneration may not facilitate such a remarkable rescue, but if this vasculature is reinforced with endothelial progenitors derived from hematopoietic stem cell populations of the bone marrow, they can make the vasculature more resistant. to degeneration and at the same time facilitate neuronal retinal survival, as well as vascular. In humans with retinal degeneration, the delay in the onset of complete retinal degeneration may provide years of additional vision. Animals treated with Lin-HCS had significant conservation of an ERG, which may be sufficient to maintain vision.

Clinically, it is widely appreciated that there may be substantial loss of photoreceptors and other neurons while still maintaining functional vision. At some point, the critical threshold is crossed and vision is lost. Since almost all human inherited retinal degenerations are early, although slow, an individual with retinal degeneration can be identified and treated intravitreally with an autologous bone marrow stem cell graft to delay degeneration of the retina and concomitant loss of vision. To enhance the targeting and incorporation of cells, the presence of activated astrocytes is desirable (Otani et al. 2002, Nat. Med. 8: 1004-1010); This can be achieved by early treatment when there is an associated gliosis, or by using a laser to stimulate local proliferation of activated astrocytes. Optionally, ex vivo transfection of the stem cells with one or more neurotrophic substances can be used before intraocular injection to enhance the rescue effect. This approach can be applied to the treatment of other visual neuronal degenerative disorders, such as glaucoma, in which there is degeneration of retinal ganglion cells.

Adult bone marrow Lin-HSC populations contain a population of EPC that can promote angiogenesis by selecting reactive astrocytes as target and incorporating into an established template without altering the structure of the retina. Lin-HSCs also provide a long-term neurotrophic rescue effect in eyes suffering from retinal degeneration. In addition, autologous, genetically modified Lin-HSC compositions containing EPC can be transplanted into ischemic or abnormally vascularized eyes and can be stably incorporated into new vessels and neuronal layers and continuously deliver therapeutic molecules locally for periods of time. prolonged Such a local supply of genes that express pharmacological agents in physiologically significant doses represents a new paradigm for treating untreatable eye diseases today.

The photoreceptors in the normal mouse retina, for example, are predominantly rods, but the outer nuclear layer observed after rescue with Lin-HCS contained predominantly cones. Most inherited human retinal degenerations occur as a result of stick-specific primary defects, and it is believed that the loss of cones is secondary to stick dysfunction, which is probably related to the loss of some trophic factor expressed by the canes.

5 Examples

Example 1. Isolation and enrichment of cells, preparation of populations A and B of murine Lin-HSC.

General procedure. All in vivo evaluations were performed according to the Guide for the Care and Use of

10 Laboratory Animals (NIH Guide to the Care and Use of Laboratory Animals), and all evaluation procedures were approved by the Animal Care and Use Committee of the Scripps Research Institute (TSRI, La Jolla, CA). Bone marrow cells were extracted from adult mice B6.129S7-Gtrosa26, Tie-2GFP, ACTbEGFP, FVB / NJ (mouse rdl / rd) or Balb / cBYJ (The Jackson Laboratory, ME).

15 Then, the monocytes were separated by density gradient separation using HISTOPAQUE® polysaccharide gradient (Sigma, St. Louis, MO) and labeled with biotin-conjugated lineage panel antibodies (CD45, CD3, Ly-6G, CD11 , TER-119, Pharmingen, San Diego, CA) for the selection of Lin- in mice. Positive lineage (Lin +) cells were separated and removed from Lin-HSC using a separation device

20 magnetic (AUTOMACS ™ classification device, Miltenyi Biotech, Auburn, CA). The resulting Lin-HSC population, which contained endothelial progenitor cells, was further characterized using a FACS ™ Calibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ) using the following antibodies: Sca-1 conjugated to PE, c-kit, KDR and CD31 (Pharmingen, San Diego, CA). Tie-2-GFP bone marrow cells were used for characterization of Tie-2.

25 To collect adult mouse endothelial cells, ACTbEGFP mouse mesenteric tissue was surgically removed and placed in collagenase (Worthington, Lakewood, NJ) to digest the tissue, followed by filtration using a 45 µm filter. The non-retained fraction was collected and incubated with endothelial growth medium (Clonetics, San Diego, CA). Endothelial characteristics were confirmed by observing the appearance of

30 morphological paving, staining with AcM in front of CD31 (Pharmingen) and examining the cultures to determine the formation of tube-like structures in the MATRIGEL ™ matrix (Beckton Dickinson, Franklin Lakes, NJ).

Population A of murine Lin-HSC. ACTbEGFP mouse bone marrow cells were removed by the general procedure described above. Lin-HSC cells were characterized by flow cytometry

35 FACS for CD31, c-kit, Sca-1, Flk-1 and Tie-2 cell surface antigen markers. The results are shown in Figure 1 (c). Approximately 81% of the Lin-HSCs showed the CD31 marker, approximately 70.5% of the Lin-HSCs showed the c-kit marker, approximately 4% of the Lin-HSCs showed the Sca-1 marker, approximately the 2.2% of the Lin-HSCs showed the Flk-1 marker and approximately 0.91% of the Lin-HSCs showed the Tie-2 marker. In contrast, the Lin + HSC that

40 isolated from these bone marrow cells had a significantly different cell marker profile (i.e., CD31: 37.4%; c-kit: 20%; Sca-1: 2.8%; Flk-: 0.05 %).

Population B of murine Lin-HSC. Bone marrow cells were extracted from Balb / C, ACTbEGFP and C3H mice by the general procedure described above. Lin-HSC cells were analyzed to determine the

45 presence of cell surface markers (Sca-1, Flk-1 / KDR, c-kit (CD117), CD34, CD31 and various integrins: a1, a2, a3, a4, a5, a6, aL, aM aV, aX , aIIb, 11, 12, 13, 14, 15 and 17). Results are shown in table 2.

Table 2. Characterization of the B-population of Lin-HSC cells

Cell dialer

LinHSC

a1

0.10

a2

17.57

a3

0.22

a4

89.39

to 5

82.47

a6

77.70

to the

62.69

A.M

35.84

aX

3.98

av

33.64

aIIb

0.25

eleven

86.26

12

49.07

13

45.70

Cell dialer

LinHSC

14

0.68

fifteen

9.44

17

11.25

CD31

 51.76

CD34

 55.83

Flk-1 / KDR

2.95

c-kit (CD117)

74.42

Sca-1

 7.54

Example 2. Intravitreal administration of cells in a murine model.

An eyelid fissure was performed on a mouse eyelid with a thin blade to expose the P2 eyeball

5 to P6. The population of HSC of negative lineage (about 105 cells in about 0.5 µL to about 1 µl of cell culture medium) was then injected intravitreally using a 33 gauge needle syringe (Hamilton, Reno, NV ).

Example 3. EPC transfection.

10 Murine Lin-HSC (population A) was transfected with DNA (SEQ ID NO: 1, FIG. 7) encoding the TrpRS T2 fragment (SEQ ID NO: 3) which also includes a His6 tail using the transfection reagent FuGENE ™ 6 (Roche, Indianapolis, IN) according to the manufacturer's protocol. Lin-HSC cells (approximately 106 cells per ml) were suspended in opti-MEM® medium (Invitrogen, Carlsbad, CA) containing stem cell factor (PeproTech,

15 Rocky Hill, NJ). The mixture of DNA (about 1 µg) and FuGENE reagent (about 3 µl) was then added and the mixtures were incubated at about 37 ° C for about 18 hours. After incubation, the cells were washed and collected. The transfection rate of this system was approximately 17% as confirmed by FACS analysis. The production of T2-TrpRS was confirmed by Western blot. The amino acid sequence of T2-TrpRS with His6 tail is

20 shows as SEQ ID NO: 2, figure 8.

Example 4. Immunohistochemistry and confocal analysis.

Mouse retinas were collected at various time points and prepared either for microscopic preparation.

25 complete or for frozen cuts. For complete microscopic preparations, the retinas were fixed with 4% paraformaldehyde and blocked in 50% fetal bovine serum (FBS) and 20% normal goat serum for one hour at room temperature. Retinas were processed to determine primary antibodies and detected with secondary antibodies. The primary antibodies used were: anti-collagen IV (Chemicon, Temecula, CA), anti-1-gal (Promega, Madison, WI), anti-GFAP (Dako Cytomation, Carpenteria, CA), anti-a-actin muscle

30 smooth (a-SMA, Dako Cytomation). Secondary antibodies used were conjugated with Alexa 488 or 594 fluorescent markers (Molecular Probes, Eugene, OR). Images were taken using an MRC 1024 confocal microscope (Bio-Rad, Hercules, CA). Three-dimensional images were created using LASERSHARP® software (Bio-Rad) to examine the three different layers of vascular development in the microscopic preparation of the entire retina. The difference in GFP pixel intensity between mice with GFP was used

35 enhanced (eGFP) and mice with GFAP / wtGFP, distinguished by confocal microscopy, to create three-dimensional images.

Example 5. Quantification assay of retinal angiogenesis in vivo in mice.

40 For the T2-TrpRS analysis, the primary and deep plexus were reconstructed from the three-dimensional images of the mouse retinas. The primary plexus was divided into two categories: normal development, or arrested vascular progression. The categories of inhibition of deep vascular development were constructed based on the percentage of vascular inhibition including the following criteria: the complete inhibition of deep plexus formation was called "Complete", the normal vascular development (including an inhibition

45 less than 25%) was called “Normal” and the rest was called “Partial.” For the rescue data of the rd / rd mouse, four separate areas of the deepest plexus were captured in each microscopic preparation of the entire retina using a lens 10x The total vasculature length for each image was calculated, added and compared between the groups. To acquire accurate information, Lin-HSC was injected into one eye and Lin + HSC was injected into the other eye of the same mouse. Non-injected control retinas from the same litter were taken.

50 Example 6. Adult murine models of retinal injury.

Scar and laser models were created using either a diode laser (150 mW, 1 second, 50 mm) or mechanically puncturing the mouse's retina with a 27-gauge needle. Five days after the injury,

55 cells were injected using the intravitreal procedure. The eyes of the mice were collected five days later. twenty-one

Example 7. Neurotrophic rescue of retinal regeneration.

Negative lineage hematopoietic stem cells derived from the adult murine bone marrow (Lin-HSC) have a vasculotrophic and neurotrophic rescue effect in a mouse model of retinal degeneration. Approximately 0.5 microliters containing approximately 105 Lin-HSC were injected into the right eyes intravitreally and were evaluated 2 months later to determine the presence of retinal vasculature and for nuclear count of the neuronal layer Approximately the same number of Lin + HSC was injected into the left eyes of the same mice as control, and were evaluated in a similar manner. As shown in Figure 9, in eyes treated with Lin-HSC, the retinal vasculature seemed almost normal, the inner nuclear layer was almost normal, and the outer nuclear layer (ONL) presented approximately 3 to approximately 4 layers. of cores. In contrast, the contralateral eye treated with Lin + HSC had a markedly atrophic middle retinal vascular layer, a completely atrophic external retinal vascular layer; the inner nuclear layer was markedly atrophic and the outer nuclear layer had completely disappeared. This was drastically illustrated in mouse 3 and mouse 5. In mouse 1, there was no rescue effect and this was true for approximately 15% of the injected mice.

When visual function was evaluated with electroretinograms (ERG), the restoration of a positive ERG was observed when both vascular and neuronal rescue were observed (mice 3 and 5). No positive ERG was observed when there was no vascular or neuronal rescue (mouse 1). This correlation between vascular and neurotrophic rescue of the eyes of the rd / rd mouse using the Lin-HSC is illustrated by a regression analysis graph shown in Figure 10. A correlation was observed between neuronal recovery (y axis) and the vascular (x axis) for the type of intermediate vasculature (r = 0.45) and for the deep vasculature (r = 0.67).

Figure 11 shows the absence of any statistically significant correlation between vascular and neuronal rescue using Lin + HSC. The vascular rescue was quantified and the data are presented in Figure 12. Data for mice at 1 month (1M), 2 months (2M) and 6 months (6M) after injection shown in Figure 12, show that the length Vascular increased significantly in eyes treated with Lin-HSC (dark bars) in relation to vascular length in untreated eyes of the same mouse (light bars), particularly at 1 month and 2 months after injection. The neurotrophic rescue effect was quantified by counting the nuclei in the internal and external nuclear layers approximately two months after the injection of Lin-HSC or Lin + HSC. The results are presented in Figure 13 and 14.

Example 8. Population of human Lin-HSC.

Bone marrow cells were removed from healthy adult human volunteers by the general procedure described above. The monocytes were then separated by density gradient separation using HISTOPAQUE® polysaccharide gradient (Sigma, St. Louis, MO). To isolate the Lin-HSC population of human bone marrow mononuclear cells, the following biotin-conjugated lineage panel antibodies were used with the magnetic separation system (AUTOMACS ™ classification device, Miltenyi Biotech, Auburn, CA): CD2, CD3, CD4, CD11a, Mac-1, CD14, CD16, CD19, CD33, CD38, CD45RA, CD64, CD68, CD86, CD235a (Pharmingen).

The population of human Lin-HSCs was further separated into two subpopulations based on CD133 expression. Cells were labeled with antibodies against CD133 conjugated with biotin and separated into subpopulations positive for CD133 and negative for CD133.

Example 9. Intravitreal administration of human and murine cells in murine models for retinal degeneration.

Mouse strains C3H / HeJ, C3SnSmn.CB17-Prkdc SCID and rd10 were used as models of retinal degeneration. The C3H / HeJ and C3SnSmn.CB17-Prkdc SCID mice (The Jackson Laboratory, Maine) were homozygous for the retinal degeneration mutation 1 (rd1), a mutation that causes the early onset of severe retinal degeneration. The mutation is located in exon 7 of the Pde6b gene that codes for the P subunit of the cGMP phosphodiesterase of the rod photoreceptors. Mutation in this gene has been found in human patients with autosomal recessive retinitis pigmentosa (RP). C3SnSmn.CB17-Prkdc SCID mice are also homozygous for spontaneous mutation of severe combined immune deficiency (Prkdc SCID) and were used for human cell transfer experiments. Retinal degeneration in rd10 mice is caused by a mutation in exon 13 of the Pde6b gene. This is also a clinically relevant RP model with retinal degeneration of posterior appearance and milder than rd1 / rd1). All valuations were performed according to the NIH Guide for the Care and Use of Laboratory Animals, and all procedures were approved by the Animal Care and Use Committee. use of animals) from the Scripps Research Institute.

An eyelid fissure was performed on a mouse eyelid with a thin blade to expose the eyeball from P2 to P6. HCS cells of negative lineage were then injected for the murine A population or the human C population (approximately 105 cells in approximately 0.5 µl approximately 1 µl cell culture medium) into the mouse intravitreally using a syringe with 33 gauge needle (Hamilton, Reno, NV). To visualize the injected human cells, the cells were labeled with dye (Cell tracker green CMFDA, Molecular Probes) before injection.

Retinas were collected at various time points and fixed with 4% paraformaldehyde (PFA) and methanol, followed by blocking in 50% FBS / 20% NGS for one hour at room temperature. To stain the retinal vasculature, retinas were incubated with anti-CD31 (Pharmingen) and anti-collagen IV (Chemicon) antibodies followed by secondary antibodies conjugated with Alexa 488 or 594 (Molecular Probes, Eugene, Oregon). Flat retinas with four radial relaxation incisions were arranged to obtain a complete microscopic preparation. Images of the vasculature were obtained in the deep or intermediate retinal vascular plexus (see Dorrell et al. 2002 Invest Ophthalmol. Vis. Sci. 43: 3500-3510) using a Radiance MP2100 confocal microscope and LASERSHARP® software (Biorad, Hercules, California). For quantification of the vasculature, four independent fields (900 µm x 900 µm) were chosen randomly from the middle part of the intermediate or deep vascular layer and the total length of the vasculature was measured using LASERPIX® analysis software (Biorad ). The total lengths of these four fields in the same plexus were used for further analysis.

Flat mounted retinas were re-embedded to obtain cuts with cryostat. Retinas were placed in 4% PFA overnight followed by incubation with 20% sucrose. Retinas were embedded in compound for optimum temperature cutting (OCT: Tissue-Tek; Sakura FineTech, Torrance, CA). The cryostat sections (10 µm) were rehydrated in PBS containing the DAPI nuclear dye (Sigma-Aldrich, St. Louis, Missouri). Images of DAPI-labeled nuclei of three different areas (280 µm width, non-skewed sample) were taken in a single section containing the optic nerve head and the entire peripheral retina using a confocal microscope. The number of nuclei located in the ONL of the three independent fields in a cut were counted and added for analysis. A simple linear regression analysis was performed to examine the relationship between the length of the vasculature in the deep plexus and the number of cell nuclei in the ONL.

After dark adaptation at night, mice were anesthetized by intraperitoneal injection of 15 µg / mg ketamine and 7 µg / mg xylazine. Electroretinograms (ERG) of the corneal surface of each eye were recorded after pupil dilation (1% atropine sulfate) using a gold ring corneal electrode together with a reference electrode in the mouth and a ground electrode in The tail. Stimuli were produced with a Grass Photic stimulator (PS33 Plus, Grass Instruments, Quincy, MA) attached to the outside of a highly reflective Ganzfeld dome. The responses of the rods to short-wavelength light flashes (Wratten 47A; Amax = 470 nm) were collected over a range of intensities of up to the maximum allowable by the photic stimulator (0.668 cd-s / m2) . The response signals were amplified (amplifier CP511 AC, Grass Instruments), digitized (PCI-1200, National Instruments, Austin, TX) and analyzed by computer. Each mouse served as its own internal control with ERGs recorded from both treated and untreated eyes. The average of up to 100 scans was performed for the weakest signals. The average responses of the untreated eye were digitally subtracted from the responses of the treated eye and this difference in the signal was used to index the functional rescue.

Microalignment analysis was used to evaluate the expression of the retinal gene selected as a target by Lin-HSC. P6 rd / rd or Lin- or CD31-HSC mice were injected. The retinas of these mice were dissected 40 days after injection in RNAse-free medium (rescue of the retinal vasculature and the photoreceptor layer is obvious at this time point after injection). A quadrant of each retina was analyzed by complete microscopic preparation to ensure that selection as a target had been achieved by normal HSCs, as well as neuronal and vasculature protection. RNA was purified from the retinas with satisfactory injections using an RNA isolation protocol in phenol / chloroform TRIzol (Life Technologies, Rockville, MD). RNA was hybridized with Affymetrix Mu74Av2 chips and gene expression was analyzed using GENESPRING® software (SiliconGenetics, Redwood City, CA). Purified human or mouse HCS were injected intravitreally into P6 mice. At P45, the retinas were dissected and assembled in fractions of 1) rescued mouse retinas, to which human HSCs were injected, 2) unrescued mouse retinas to which human HSCs were injected, and 3) retinas of rescued mice that were injected with mouse HSC for RNA purification and hybridization with human-specific Affymetrix U133A chips. GENESPRING® software was used to identify genes that were expressed above the background and with greater expression in retinas rescued with human HSCs. The expression profiles of the pair of probes for each of these genes were then analyzed individually and compared with a model of normal human U133A microalignment experiments using dChip to determine the specific hybridization of the human species and to eliminate false positives due to cross species hybridization.

Figure 21 illustrates flow cytometry data comparing the expression of CD31 surface antigens and alpha 6 integrin in CD133 (CD133 +) and CD133 positive (CD133-) human Lin-HSC populations. The left panels show flow cytometry scatter plots. The central and right panels are histograms that show the level of expression of specific antibodies in the cell population. The Y axis represents the number of events and the X axis shows the signal strength. The histograms depicted in contour are IgG isotype control antibodies that show the level of non-specific background staining. Filled histograms show the level of expression of specific antibodies in the cell population. A filled histogram shifted to the right of the histogram depicted in contour (control) represents an increase in fluorescent signal and antibody expression above the background level. The comparison of the position of the peaks of the stuffed histograms between the two cell populations represents the difference in the expression of proteins in the cells. For example, CD31 is expressed above the background in both CD133 + and CD133- cells; however, there are more cells that express lower levels of CD31 in the population of CD133 + cells than in the CD133- population. From these data it is evident that the expression of CD31 varies between the two populations and that the expression of the integrin alfa 6 is mostly limited to the cells in the Lin- population, and therefore can serve as a cell marker with vasculotrophic and neurotrophic rescue function.

When the subpopulation of Lin-HSC positive for CD133 and negative for CD133 was injected intravitreally into the eyes of neonatal SCID mice, the highest degree of incorporation into the developing vasculature was observed for the subpopulation negative for CD133, which expresses both antigens of surface CD31 and integrin a6 (see Figure 21, bottom). The positive subpopulation for CD133, which does not express CD31 or integrin a6 (Figure 21, upper part) seems to select sites of neovascularization directed by peripheral ischemia, but not when injected into eyes undergoing angiogenesis.

Rescued and un rescued retinas were analyzed by immunohistochemistry with antibodies specific for cone or rod opsin. The same eyes used for the ERG records presented in Figure 17 were analyzed to detect the opsin of cones or rods. In wild-type mouse retinas, less than 5% of the photoreceptors present are cones (Soucy et al. 1998, Neuron 21: 481-493) and immunohistochemical staining patterns observed with red / green cones opsin as shown in figure 25 (A) or red rhodopsin as shown in figure 25 (B), they agreed with this percentage of cone cells. Dr. Robert Molday of the University of British Columbia provided antibodies specific for red rhodopsin (rho4D2) and were used as described previously (Hicks et al. 1986, Exp. Eye Res. 42: 55-71). Rabbit antibodies specific for Chemicon red / green cone opsin (AB5405) were purchased and used according to the manufacturer's instructions.

Example 10. Intravitreal administration of murine cells in murine models for oxygen-induced retinal degeneration.

Newborn wild-type C57B16 mice were exposed to hyperoxia (75% oxygen) between days P7 to P12 postnatal in a model of oxygen-induced retinal degeneration (OIR). Figure 22 illustrates normal postnatal vascular development in C57B 16 mice from P0 to P30. At P0, only incipient surface vessels can be observed around the optical disc. During the following days, the primary surface network extends towards the periphery, reaching the distant periphery towards day P10. Between P7 and P12, the secondary (deep) plexus develops. Towards P17, a wide surface and deep network is present (figure 22, insertions). In the following days, remodeling occurs along with the development of the tertiary (intermediate) layer of vessels until the adult structure is reached approximately at P21.

In contrast, in the OIR model described herein, after exposure to 75% oxygen in P7-P12, the normal sequence of events is seriously disturbed (Figure 23). Adult murine Lin-HSC populations were injected intravitreally into P3 in one eye of a mouse that subsequently underwent OIR, negative cells for CD31 or PBS were injected as control. Figure 24 illustrates that Lin-HSC populations can reverse the degenerative effects of high oxygen levels in the developing mouse retina. Fully developed superficial and deep retinal vasculature at P 17 was observed in the treated eyes, while the control eyes showed large avascular areas with virtually no deep vessels (Figure 24). Approximately 100 eyes of mice were observed in the OIR model. Normal vascularization was observed in 58% of eyes treated with Lin-HSC populations, compared with 12% of control eyes treated with CD31-cells and 3% of control eyes treated with PBS.

Example 11. Isolation of myeloid bone marrow cells from the murine bone marrow by CD44 selection.

Bone marrow cells were removed from adult mice (The Jackson Laboratory, ME). The entire bone marrow was treated with an antibody against murine CD44 and flow cytometry was used to isolate cells expressing CD44 from the bone marrow. The antibody cells were separated and stored in a buffer solution for future use. A population of cells that did not significantly express CD44 (CD44loBM) was also isolated.

Example 12. Isolation of myeloid bone marrow cells from the murine bone marrow by CD44 selection.

facing CD44, as described in example 11. A population of myeloid-type bone marrow cells was isolated that was CD44hi and CD11b +, which had similar activity characteristics to those of the CD44hi population isolated in example 11 using CD44. A population of CD44 or CD11b- was also isolated, which was found to be inactive.

5 Example 13. Characterization of MLBM cell populations.

Although the role of CD44 in this context is unclear, it is possible that this receptor mediates cell survival, cell migration and / or cell differentiation in vitreous humor rich in hyaluronic acid after injection

10 cells in the eye. Different populations of CD44hi cells (ie MLBM) and CD44lo were present in the unfractionated mouse bone marrow. The MLBM cell population represents 76% of the Lin-population used in the previous examples, while only about 37% and 4%, respectively, of the Lin + and CD31- / CD34- / CD11b- cell populations The bone marrow expressed CD44 (Figure 26). Therefore, there is an excellent correlation between CD44 expression and vasculotrophic activities and

The neurotrophic observed in these three populations, that is, the Lin- cells were the most effective while the CD31- / CD34- / CD11b- cells were systematically the least effective. Using a panel of specific lineage antibodies, it was determined that most CD44hi cells had strongly myeloid characteristics (Figure 27). Similarly, almost all CD44hi bone marrow cells are also CD11b + (Figure 27).

20 MLBM positively selected using an antibody against CD11b in example 12 (CD44hi CD11b +) gave similar activity results to those obtained with MLBM isolated using antibody selection against CD44 in vascular targeting experiments.

25 Table 3 below shows the characteristics of cell surface antigens of the MLBM cell population of example 12 and of the CD44 or CD11b + cells isolated in example 12. In Table 3, a greater number of positive signs (+ ) indicates relatively higher antigen expression. A negative sign (-) indicates that no expression was detected.

30 Table 3

CD44hi / CD11b + CD44lo / CD11b-CD11a +++ + CD31 + ++ CD34 + -alfa 6 ++ -KDR + -Sca-1 + + c-Kit + -CD115 + -CD45RB220 + ++ TER119 - +++ Ly6G & C (GR-1) +++ -Ly6G +++ -

Example 14. Vasculotrophic and neurotrophic effects of the MLBM cell population

The population of MLBM cells of Example 11 retained the properties of Lin-cells in terms of vascular targeting and vasculotrophic and neurotrophic effects, while CD44loBM cells showed little or no activity. Vascular targeting activity was demonstrated by injecting cells from a population of GFP + MLBM cells intravitreally into mice on postnatal day 7 (P7) and analyzing the retinas in P14. After marking the blood vessels with GS isolectin, it was observed that GFP + cells selected as

40 target the retinal vasculature and adopted a perivascular location, without evidence of incorporation. These events were common when MLBM were used, but infrequent or absent in CD44loBM treated eyes (Figure 28).

Vasculotrophic and neurotrophic activity of the MLBM cell population of Example 11 was evaluated using a

45 mouse model of retinal degeneration as described above for Lin-HSC. The rd1 / rd1 mouse shows characteristic features of degenerative retinal disease including death of photoreceptors and atrophy of the deep retinal vasculature. As described above, the Lin-HSC bone marrow cells preserved the vasculature of the deep retina and partially rescued the photoreceptors. The population of MLBM cells performs the same function (figure 29).

50 The oxygen-induced retinopathy model shares characteristics with retinopathy of prematurity. The pathology associated with this model is significantly reduced when the eyes are treated with cells from the MLBM cell population. The effects of the cells of the MLBM cell population in this model were similar to those observed using Lin-HCS described above. The eyes treated with cells from the MLBM cell population showed a significant reduction in the two parameters used to quantify the degree of pathology in this model: the area of vascular obliteration and the area of neovascular glomeruli. In contrast, eyes treated with CD4410BM cells showed no improvement with respect to eyes treated with vehicle controls (Figure 30).

In addition to selecting the retinal vasculature as a target, the cells of the MLBM cell population differentiate into macrophage-like cells (F4 / 80 +), penetrate the retina and adopt a position closely opposite to the retinal pigment epithelium ( RPE) This location facilitates the effects of vascular rescue and observed photoreceptors of the cells of the MLBM cell population. In addition, once in place near the RPE, the cells of the MLBM cell population produce vascular endothelial growth factor (VEGF), as demonstrated by the injection of cells from a population of MLBM cells derived from a VEGF- mouse GFP, in which the green fluorescent protein (GFP) is expressed after activation of the VEGF gene (Figure 31). Therefore, the cells of the MLBM cell population appear to be in an "activated" state of VEGF. The introduced cells of the MLBM cell population appear to recruit endogenous cells of the same type, since both GFP + (introduced) and GFP- (endogenous) cells were observed in the RPE region. This location has been observed in wild-type mice during the normal retinal vascular development, in retinas rescued in the rd1 * rd1 mouse and in the oxygen-induced retinopathy model.

Similar vascular targeting results were found for the MLBM cell population of the example

12. Figure 32 shows that towards P20, the CD44hi CD11b cells of example 12 (green color) specifically selected the vasculature (red color) when injected into P2, in a manner similar to the CD44-high population of the example 11. Figure 33 shows that the CD44 or CD11b- of Example 12 did not specifically target the vasculature.

The MLBM cell population provides an effective and versatile treatment for similar ischemic and ocular retinopathic diseases. The cells are easily isolated from autologous bone marrow, thus minimizing the possible immunogenicity often observed in cell-based therapies. Long-term follow-up (up to six months) revealed only occasional rosettes and histological preservation of the neural retina in eyes that were injected with negative lineage cells. In addition, the population of MLBM cells can be transfected with genes useful to deliver functional genes to the retina.

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Claims (11)

1. Use of cells in the preparation of a pharmaceutical composition for the treatment of a mammal that is at risk of developing or suffering from a retinopathic disease, in particular a retinopathy of prematurity, in which:

(to)

the cells are a population of myeloid, vasculotropic, negative lineage bone marrow cells, in which the cells express the CD44 and CD11b antigens;

(b)

the cells have been transfected with a nucleic acid encoding an angiostatic fragment of tryptophanyl-tRNA synthetase (Trp-RS);

(C)

The pharmaceutical composition is for the administration of said cells to the mammalian retina in an amount effective to favor the physiological revascularization of the damaged areas of the retina and to improve the lesion to the retina caused by the disease.

2.

Use according to claim 1, wherein the angiostatic TrpRS fragment is T2-TrpRS (SEQ ID NO: 3) or T2-TrpRS-GD (SEQ ID NO: 4).

3.

Use according to claim 1, wherein the cells comprise hematopoietic stem cells and endothelial progenitor cells.

Four.

Use according to claim 1, wherein the cells are produced by a method comprising isolating bone marrow from a mammal and positively selecting bone marrow cells that immunoreact with an antibody selected from the group consisting of anti-CD44, anti- CD11b and a combination thereof.

5.

Use according to claim 1, wherein the mammal is a human being.

6.

Use according to claim 1, wherein the mammal is a lactating mammal.

7.

Use according to claim 6, wherein the nursing mammal has been exposed to hyper-toxic conditions.

8.

Use according to claim 1, wherein the pharmaceutical composition is for the administration of the cells by intraocular injection.

9.

Use according to claim 1, wherein the cells are autologous to the mammal being treated.

10.

Use according to claim 1, wherein the pharmaceutical composition is for the administration of the cells before the onset of disease symptoms.

eleven.

Use according to claim 1, wherein the pharmaceutical composition is for the administration of the cells before exposing the mammal to hyper-toxic conditions.